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US7790377B2 - Compounds that act to modulate insect growth and methods and systems for identifying such compounds - Google Patents

Compounds that act to modulate insect growth and methods and systems for identifying such compounds Download PDF

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US7790377B2
US7790377B2 US10/929,090 US92909004A US7790377B2 US 7790377 B2 US7790377 B2 US 7790377B2 US 92909004 A US92909004 A US 92909004A US 7790377 B2 US7790377 B2 US 7790377B2
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ecr
fxr
activity
ecdysteroid
receptor
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US20050049230A1 (en
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Vincent C. Henrich
Cary Alan Weinberger
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University of North Carolina at Greensboro
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University of North Carolina at Greensboro
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Priority to US11/543,682 priority patent/US8637237B2/en
Priority to US12/731,810 priority patent/US20100240056A1/en
Priority to US12/843,485 priority patent/US20110207707A1/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects

Definitions

  • the present invention relates to compounds that act to modulate insect growth and methods and systems for identifying such compounds.
  • Insect development appears to be driven by the action of at least two hormone classes, the ecdysteroids and the juvenile hormones (JHs, juvenoids) (Riddiford, L. M., 1994 , Adv. Insect Physiol., 24:213-274; Gilbert, L. I., et al., 2000 , Insect Biochem. Mol. Biol., 30:617-644; Thummel, C. S., 2002 , Insect Biochem. Mol. Biol., 32:113-120). It appears that ecdysteroids are responsible for initiating metamorphosis, and in some insects, regulating adult fertility.
  • JHs juvenile hormones
  • JH appears to be required for reproductive processes such as adult female vitellogenesis (Wyatt, G. R., and K. G. Davey, 1996 , Adv. Insect Physiol., 26:1-15). Also, the simultaneous presence of ecdysteroids and juvenile hormone (JH) leads to larval-larval molting.
  • EcR ecdysone receptor
  • USP Ultraspiracle
  • DHR38 a second receptor, as a heterodimeric partner of USP has been shown to mediate ecdysteroid responses, but the mode of action is not classical, in that it does not involve direct binding of the ecdysteroid to either DHR38 or USP (Baker et al, 2003 , Cell 113:731-742).
  • EcR structurally resembles the vertebrate famesoid X-activated receptor (FXR) (Forman, B. M., et al., 1995 , Cell 81:687-695).
  • FXR is a member of the steroid receptor family that includes receptors for glucocorticoids, estrogen, vitamins A and D, thyroid hormones, and fatty acids.
  • USP may be the insect orthologue of the vertebrate retinoid X receptor (RXR) (Oro, A. E., et al., 1990 , Nature 347:298-301).
  • Embodiments of the present invention provide compounds that act to modulate insect growth and methods and systems for identifying such compounds.
  • the present invention may be embodied in a variety of ways.
  • the present invention comprises a method for testing the ability of a compound to act as a modulator of insect growth comprising determining whether the compound increases FXR-mediated transcription and/or EcR-mediated transcription.
  • the method comprises a cell based assay.
  • the method may comprise transfecting a cell with DNA comprising a functional isoform of the ecdysone receptor (EcR) or the farnesoid X-activated receptor (FXR).
  • EcR ecdysone receptor
  • FXR farnesoid X-activated receptor
  • a cell that does not include EcR, FXR, or a binding partner for EcR or FXR is used for the assay so that there may be minimal background due to endogenous activation via EcR or FXR.
  • the method may further comprise cotransfecting the cell with a DNA construct comprising a functional heterologous binding partner for either the EcR or FXR.
  • Increases in EcR-mediated transcription or FXR-mediated transcription may be measured in a variety of ways.
  • DNA comprising a gene linked to a hormone response element (HRE) that recognizes an activated EcR complex or an activated FXR complex may be transfected into the cell.
  • the gene linked to the HRE may be a reporter gene that is easily measured, as for example, the chloramphenicol acetyltransferace (CAT) gene.
  • CAT chloramphenicol acetyltransferace
  • the increase in reporter gene activity may be used to quantify the activity of the test compound as a potential insecticide, where the ability of a compound to act as an insecticide is positively correlated with activation of HRE mediated transcription of the reporter gene.
  • the present invention also comprises a method that may be used to screen for insecticides in the environment.
  • the present invention comprises a method for in situ testing for the presence of compound having the ability to modulate insect growth comprising transfecting a cell in an organism with DNA that encodes a functional isoform of the farnesoid X-activated receptor (FXR) and/or the ecdysone receptor (EcR).
  • the cell may also be transfected with a DNA encoding a functional RXR or USP isoform.
  • the cell may comprise a DNA comprising a hormone response element (HRE) linked to a reporter gene.
  • HRE hormone response element
  • the cell may be exposed to an ecdysteroid. The organism may then be exposed to the compound to be tested to determine the effect of the compound on FXR-activated transcription and/or EcR-activated transcription.
  • the present invention further comprises assay systems.
  • the present invention may comprise an assay system for the identification of compounds having the ability to act as a modulator of insect growth comprising a host cell transfected with an exogenous DNA construct comprising sequences encoding a functional isoform of the ecdysone receptor (EcR) or the famesoid receptor (FXR); an exogenous DNA construct comprising sequences encoding a functional heterologous binding partner for EcR or FXR, wherein the heterologous binding partner forms a complex with either EcR or FXR, and wherein the complex binds to a hormone responsive element (HRE) on DNA to activate gene transcription; and DNA comprising a reporter construct comprising an EcR or FXR activated hormone response element (HRE) linked to a gene.
  • an assay system for the identification of compounds having the ability to act as a modulator of insect growth comprising a host cell transfected with an exogenous DNA construct comprising sequences encoding a functional iso
  • the present invention also comprises compounds that act as insecticides by their ability to inhibit insect growth, reproduction, and/or morphogenesis.
  • the present invention comprises a composition for use as an insecticide comprising a compound that increases FXR-mediated transcription and/or EcR-mediated transcription mixed with a suitable carrier for application to plants.
  • the present invention may comprise a use for compounds identified using the methods and systems of the invention as insecticides and/or modulators of insect growth.
  • the present invention comprises methods and systems to identify natural compounds derived from plants that have the ability to increase FXR-mediated transcription and/or EcR-mediated transcription and thus, may be potential insecticides.
  • Such natural compounds are potentially non-toxic insecticides; some of the compounds may even form part of the human diet.
  • the present invention may also provide for the discovery and/or refinement of ecdysteroid agonists and antagonists which exhibit a high potency for a targeted pest species, while exerting little impact on colocalized nonpest insects.
  • the diversity of chemical structures associated with ecdysteroid agonist and antagonist activity indicates that there may be numerous potential agents having insecticidal or growth modifying activities that may be characterized using the systems and methods of the present invention. Because many of the compounds of the present invention are present in human dietary sources, they may be particularly useful for the control of insects that act as vectors or predators of animal and human populations. Thus, using the compounds, methods, and systems of the present invention, it may be possible to develop strategies for refining the effectiveness of existing insecticides as well as discovering new ones.
  • plants produce phytoecdysteroids and nonsteroidal agonists/antagonists
  • ecdysteroids and their receptor may be implicated in adult insect reproductive development and physiological processes.
  • the compounds of the present invention may be potentially useful for controlling adult stage as well as developing pests.
  • FIG. 1 shows a schematic diagrams of cell-based assay systems for detecting insecticides in accordance with example embodiments of the present invention where ( 1 A) showns activation of EcR-mediated transcription potentiated by an ecdysteroid by compound X, and ( 1 B) shows activation of FXR-mediated transcription by compound X.
  • FIG. 2 shows the potentiating effects of juvenile hormone III (JHIII) dosage on the muristerone-A (murA) induced response mediated by a GRdEcR chimera and mouse RXR (mRXR) using a chloramphenical acetyltransferease (CAT) reporter construct (EcRE) 5 - ⁇ MTV-CAT in mammalian Chinese Hamster Ovary (CHO) cells in accordance with example embodiments of the present invention.
  • Sets 1, 2 and 3 correspond to 0, 0.01 and 0.1 ⁇ M murA.
  • FIG. 3 shows the ecdysteroid response and potentiation effects of JHIII (log fold induction based on normalized activity in relative light units (RLUs)) using a luciferase reporter gene (EcRE) 5 - ⁇ MTV-LUC in CHO cells, and measured for various EcR combinations and mouse RXR (mRXR) in accordance with example embodiments of the present invention, wherein the constructs used were as follows: ( 3 A) VP16dEcR/mRXR at 0.01 ⁇ M murA; ( 3 B) VP16dEcR/mRXR at 0.1 ⁇ M murA; ( 3 C) VP16dEcR/VP16CfUSP at 0.1 ⁇ M murA, ( 3 D) EcRA/mRXR at 1 ⁇ M murA; ( 3 E) EcRB1/mRXR at 0.1 ⁇ M; ( 3 F) EcRB2/mRXR at 0.1 ⁇ M murA.
  • EcRE
  • FIG. 4 shows the effects of murA, 20E, and JHIII on RLU activity induced by (EcRE) 5 - ⁇ MTV-LUC in CHO cells cotransfected with Drosophila EcR isoforms and VP16CfUSP in accordance with example embodiments of the present invention where sets 1, 2, and 3 in the figure correspond to EcRA, EcRB1 and EcRB2, respectively.
  • FIG. 5 shows the response of various combinations of nuclear receptors and their binding partners to muristerone A and JHIII in CHO cells in accordance with example embodiments of the present invention, where EcR refers to VP16dEcR, USP refers to VP16CfUSP, and FXR and RXR refer to natural mammalian forms.
  • FIG. 6 shows that FXR may be activated by sesquiterpene metabolites of farnesyl diphosphate in accordance with example embodiments of the present invention wherein the relative efficacy of each isoprenoid as an inducer of FXR-dependent transcription is displayed.
  • FIG. 7 shows molecular structures of natural and synthetic juvenile hormone agonists with FXR effector activities in accordance with example embodiments of the present invention including: ( 7 A) farnesol-like activators; ( 7 B) juvenile hormone agonists from plants; and ( 7 C) methylenedioxyphenyl-like chemicals with JH activity.
  • FIG. 8 shows FXR activation profiles for plant secondary metabolites and congeners in accordance with example embodiments of the present invention, wherein the following compounds were tested for FXR activation:
  • TTO tea tree oil
  • 8 B coffee diterpenes cafestol acetate and kahweol acetate;
  • 8 C cucurbitacin D (cuc D) (1 ⁇ M) added to cells with, or without, farnesol (45 ⁇ M) or chenodeoxycholic acid (CDCA) (40 ⁇ M);
  • FIG. 9 shows FXR-mediated transcription may be increased by phenylpyrazole insecticides in accordance with an example embodiment of the present invention, wherein increasing doses of fipronil, chlorfenapyr (pirate), and imidacloprid were tested as indicated for the ability to increase FXR-mediated transcription.
  • FIG. 10 shows that FXR-mediated transcription may be inhibited or stimulated by metabolites or analogues of the JH antagonist precocene in accordance with example embodiments of the present invention wherein: ( 10 A) shows TLC separation of precocene isoforms and comparison of FXR effector activities; ( 10 B) shows that microsome-treated precocene I generates FXR antagonists; ( 10 C) shows molecular structures of precocene-like analogs that increase or inhibit FXR-dependent activity; ( 10 D) inhibition of farnesoid (F)-induced FXR-dependent transcription mediated by esculetin (E); ( 10 E) shows that FXR-mediated transcription is inhibited by ubiquinone-1, wherein the indicated doses of ubiquinone-1 were added to CHO cells transfected with plasmid DNAs to measure agonist activity and identical doses were added with 30 ⁇ M farnesol (F) to measure antagonist activity; and ( 10 F) shows that
  • FIG. 11 shows that transcriptional activity programmed by muristerone-primed ecdysone receptors may be potentiated by juvenile hormones and insecticides in accordance with example embodiments of the present invention
  • FIG. 11 A shows potentiation of ecdysone receptor activity by JH III, where muristerone A (MUR) was added at the indicated doses in ethanol vehicle, and increasing amounts of JH III (12, 25, or 50 ⁇ M) were added (underlying triangles), and numbers over bars indicate the ratio of the GEcEc-dependent activity produced by 50 ⁇ M JH III in the presence of the indicated dose of MUR to the activity produced by MUR alone
  • ( 11 B) shows that JH III activity may require both EcR and RXR
  • ( 11 C) shows that the JH agonist juvocimene from basil may be an EcR effector molecule where muristerone A (0.2 ⁇ M) was added alone or with 10 or 20 ⁇ M juvocimene (J) to GEcEc-
  • FIG. 12 shows that EcR and FXR ligand binding domains may mediate the transcriptional effects of juvenile hormones and insecticides in accordance with an embodiment of the present invention
  • 12 A shows GGEc chimeric receptor activation in the presence or absence of 0.2 ⁇ M muristerone A (mur) by farnesol (farn) (25 ⁇ M) and its nerolidol isomer
  • 12 B shows that juvocimene may potentiate muristerone-primed GEcEc and GGEc-dependent activity
  • ( 12 C) shows that GGEc may mediate the transcriptional effects of the FXR effector ubiquinone-2 (U2)
  • 12 D shows that GGF chimeric receptor-dependent activation is increased by RXR where CHO cells were transfected with the indicated combinations of FXR- or RXR-expressing plasmid DNAs and chenodeoxycholic acid (CDCA) was added at a final dose of 40 ⁇ M (+) or is absent
  • FIG. 13 shows the molecular structures of juvenile hormones isolated from insects, where the chemical formulas for the “naturally-occurring” juvenile hormones JH 0, JH I, JH II, and JH III are compared with the synthetic juvenoid ZR354, in accordance with an example embodiment of the present invention.
  • Embodiments of the present invention provide compounds that function as insect growth modulators and/or insecticides.
  • the present invention recognizes that there are nuclear receptor-gated signaling pathways in insects and mammals that may uniformly respond to a broad range of natural and synthetic insecticides.
  • the compounds of the present invention may target the ecdysone receptor (EcR) and/or the famesoid X-activated receptor (FXR) to act as insect growth regulators and/or to interfere with insect development.
  • EcR ecdysone receptor
  • FXR famesoid X-activated receptor
  • the present invention provides and methods and systems for the identification of compounds that act as insecticides or modulators of insect growth and/or development. Bioassays assembled using these receptors may be employed to guide the rational design of novel chemicals that specifically target insect pests.
  • compositions comprising compounds that function as insect growth regulators for the control of pests.
  • the compounds used as growth regulators may be natural compounds, isolated from everyday plants such as sesame seeds, hops, coffee, and bergamot oil found in tea.
  • the present invention may be embodied in a variety of ways.
  • the present invention comprises a method for testing the ability of a compound to act as a modulator of insect growth comprising determining whether the compound increases famesoid X-activated receptor (FXR)-mediated transcription and/or ecdysone receptor (EcR)-mediated transcription.
  • FXR famesoid X-activated receptor
  • EcR ecdysone receptor
  • an increase in transcription comprises an increase from a measurable basal level to a higher level.
  • an increase in transcription may comprise an increase from an undetectable level to a measurable level.
  • an increase in EcR mediated transcription comprises potentiation of hormone activated EcR mediated transcription.
  • the hormone may comprise an ecdysteroid.
  • the hormone may comprise muristerone A (murA) or 20-hydroxyecdysone (20E).
  • the method may comprise a cell based assay.
  • the method may comprise the steps of: (a) transfecting isolated cells with DNA comprising sequences that encode a functional isoform of the ecdysone receptor (EcR) or the farnesoid receptor (FXR); (b) cotransfecting the cell with: (i) DNA comprising sequences that encode a functional heterologous binding partner for (a), wherein said heterologous binding partner complexes with either EcR or FXR, and wherein the complex binds to a hormone responsive element (HRE) on DNA to activate gene transcription; and (ii) DNA comprising the reporter construct comprising a hormone response element (HRE) linked to a gene; (c) adding the compound to be tested; and (d) measuring an increase in the synthesis of the reporter gene protein.
  • an EcR modulator may be added to the cell such that potentiation of EcR activity may be measured.
  • the present invention further comprises assay systems.
  • the present invention may comprise an assay system for the identification of compounds having the ability to act as a modulator of insect growth comprising a host cell transfected with an exogenous DNA construct comprising sequences encoding a functional isoform of the ecdysone receptor (EcR) or the famesoid receptor (FXR); an exogenous DNA construct comprising sequences encoding a functional heterologous binding partner for EcR or FXR, wherein the heterologous binding partner forms a complex with either EcR or FXR, and wherein the complex binds to a hormone responsive element (HRE) on DNA to activate gene transcription; and DNA comprising a reporter construct comprising an EcR or FXR activated hormone response element (HRE) linked to a gene.
  • an assay system for the identification of compounds having the ability to act as a modulator of insect growth comprising a host cell transfected with an exogenous DNA construct comprising sequences encoding a functional iso
  • the increase in reporter gene activity may be used to quantify the ability of the test compound to increase EcR-mediated transcription or FXR-mediated transcription.
  • the ability of the test compound to increase EcR-mediated transcription or FXR-mediated transcription may correlated to the potential insecticidal activity of the test compound.
  • transcription of a gene that is downstream of a hormone response element (HRE) that is activated by FXR and/or EcR may be measured directly.
  • a reporter gene comprising an HRE may be exogenous to the cell and added by transfection.
  • transcriptional levels of an endogenous gene known to be activated by FXR or EcR may be measured.
  • transcription may be measured by quantifying mRNA specific to a gene that comprises a promoter that includes an FXR HRE.
  • transcription may be measured by quantifying enzyme activity specific to a protein encoded by a gene that comprises a promoter that includes an EcR HRE.
  • the actual amount of protein translated from a gene comprising a HRE may be measured by techniques known in the art such as sodium dodecyl sufate polyacrylamide gel electrophoresis (SDS-PAGE), or binding of an antibody specific for the translated gene product.
  • SDS-PAGE sodium dodecyl sufate polyacrylamide gel electrophoresis
  • Other methods to quantify gene transcription e.g., quantitative PCR amplification of cDNA, RNA hybridization, and the like may also be used.
  • the heterologous binding partner may vary depending upon the DNA-receptor (e.g., FXR or EcR) being used.
  • the heterologous binding partner may comprise Ultraspiracle (USP).
  • the heterologous binding partner may comprise vertebrate retinoid X receptor (RXR).
  • the EcR modulator may be one of several compounds known to interact with EcR and its heterologous binding partner.
  • the EcR modulator may bind to the ligand binding domain (LBD) of EcR.
  • the EcR modulator may bind to other portions of the EcR polypeptide.
  • the EcR modulator may comprise an ecdysteroid.
  • the EcR modulator may comprise muristerone A (murA).
  • the EcR modulator may comprise 20-hydroxyecdysone (20E).
  • the modulator may be added to the assay system as a polypeptide.
  • the modulator may comprise ponasterone A, 3-dehydro-20-hydroxyecdysone, ecdysone, makisterone A, nonsteroidal agonist including RH5849, RH59992, and the like.
  • the EcR modulator may be transfected into the cell as a DNA construct.
  • the methods and assay systems of the present invention may be designed to quantify and/or optimize the ability of compounds to interact with various isoforms of the nuclear receptor system.
  • the isoforms of EcR comprise the Drosophila EcRA, EcRB1, or EcRB2 isoforms.
  • the methods and assay systems of the present invention may employ a cell-based assay that is designed to have minimal background due to endogenous ecdysteroids.
  • the isolated cells used for transfection may comprise mammalian cells.
  • the isolated cells may comprise insect cells that do not have a EcR or that contain a non-functional EcR.
  • insect cells that comprise EcR “knock-outs,” in that the gene for EcR has been mutated to silence expression may be used.
  • the reporter construct may comprise a promoter having multiple hormone response elements (HREs) linked to a gene encoding a detectable gene product.
  • the hormone response elements may recognize activated FXR and/or activated EcR.
  • the response elements that may be used include the heat shock protein 27 (hsp 27) EcRE (Riddihough and Pelham, 1987 , EMBO J, 6:3729-3734) or other elements such as a palindromic sequence separated by a single nucleotide (PAL1) or a direct repeat separated by four nucleotides (DR4) or five nucleotides (DR5) (e.g., Vogtli et al., 1998 , Nuc.
  • PAL1 heat shock protein 27
  • DR4 direct repeat separated by four nucleotides
  • DR5 five nucleotides
  • the reporter gene may comprise chloramphenicol acetyltransferase (CAT).
  • CAT chloramphenicol acetyltransferase
  • the reporter gene may comprise luciferase (LUC).
  • LOC luciferase
  • the reporter gene may comprise green fluorescent protein (GFP).
  • the methods and assay systems of the present invention may comprise chimeric molecular substrates that allow for the analysis of structural and functional aspects of proteins involved in the modulation of insect growth and/or development. By using such chimeric molecular substrates, compounds that are targeted to specific portions of the receptor and/or its binding partner may be developed.
  • different hormone response elements may be employed.
  • the hormone response element may comprise an EcR response element, such as one found in the gene that encodes heat shock protein 27 (hsp27).
  • the hormone response element may comprise a FXR response element such as that found in the ileal bile acid binding protein gene (Grober, et al., 1999 , J. Biol. Chem., 274:29749-29754).
  • the response element may be any nucleic acid sequence that responds to FXR or EcR to stimulate gene transcription.
  • site-directed mutagenesis or randomly generated mutations within the EcR or FXR LBD may be recovered.
  • Such mutations may include sequences that enhance the induced responsiveness of the cell culture system, and/or reduce non-induced transcription levels while maintaining or increasing induced transcriptional activity, and/or change the specificity or induction properties of the receptor for the purposes of screening a subclass or potential insecticide compounds.
  • the methods and assay systems of the present invention may use DNA constructs isolated from various species to develop species-specific insecticides.
  • USP or EcR from various species may have different activities.
  • the FXR or EcR constructs and/or their respective heterologous binding partners may comprise species-specific constructs.
  • the DNA encoding the ecdysone receptor (EcR) or the farnesoid X-activated receptor (FXR) may comprise a chimera of DNA from different insect or vertebrate sources.
  • the chimera may comprise a mammalian activating domain.
  • the chimera may comprise a mammalian DNA binding domain (DBD) and/or a mammalian ligand binding domain (LBD).
  • the mammalian receptor domains e.g., activating, DBD, or LBD
  • the chimera may comprise an EcR-activating domain.
  • the chimera comprises an EcR DNA binding domain (DBD) and/or an EcR ligand binding domain (LBD).
  • the EcR sequences may be derived from an arthropod.
  • the EcR sequences may be derived from Drosophila melanogaster .
  • the chimera may comprise a viral protein-16 (VP16) activating domain from pseudorabies virus.
  • the chimera may comprise Chironomus tentans (lower Diptera ) (Cf) USP LBD, such as utilized in the VP16CfUSP construct.
  • portions of the activating domain, DNA-binding domain, and hinge region may be deleted from either the natural EcR or USP receptor.
  • one of three characterized mammalian (e.g., mouse, rat, or human) RXRs may be used as the heterologous binding partner.
  • LBDs Manduca sexta (Lepidoptera), Locusta migratoria (Orthoptera), Heliothis virescens (Leptidoptera), Apis mellifera (Hymenoptera), Aedes aegypti (lower Diptera), and Tenebrio molitor (Coleoptera) may also be employed.
  • the present invention also comprises a method that may be used to screen for insecticides in the environment.
  • the present invention comprises a method for in situ testing for the presence of compound having the ability to modulate insect growth comprising the steps of:
  • the organism comprises an insect.
  • the organism may comprise a transgenic Drosophila engineered to express heterologous EcR/USP peptides.
  • the reporter construct may comprise a promoter comprising multiple hormone response elements (HRES) linked to a gene encoding a detectable gene product.
  • HRES hormone response elements
  • the reporter gene may comprise luciferase (LUC) or green fluorescent protein (GFP).
  • hormone response elements may be employed.
  • the hormone response element comprises an EcR response element.
  • the hormone response element may comprise a FXR response element.
  • the heterologous binding partner may vary depending upon the DNA-receptor (e.g., FXR or EcR) being used.
  • the heterologous binding partner may comprise Ultraspiracle (USP).
  • the heterologous binding partner may comprise vertebrate retinoid X receptor (RXR).
  • the EcR modulator may be one of several compounds known to interact with EcR and its heterologous binding partner.
  • the EcR modulator may bind to the ligand binding domain (LBD) of EcR.
  • the EcR modulator may bind to other portions of the EcR polypeptide.
  • the EcR modulator may comprise an ecdysteroid.
  • the EcR modulator may comprise muristerone A (murA).
  • the EcR modulator may comprise or 20-hydroxyecdysone (20E).
  • the modulator may comprise ponasterone A, 3-dehydro-20-hydroxyecdysone, ecdysone, makisterone A, nonsteroidal agonist including RH5849, RH59992, and the like.
  • the modulator may be added to the assay system as a polypeptide.
  • the EcR modulator may be transfected into the cell as a DNA construct.
  • the assay system may be designed to quantify and/or optimize the ability of compounds to interact with various isoforms of the nuclear receptor system.
  • the isoforms of EcR comprise the Drosophila EcRA, EcRB1, or EcRB2 isoforms.
  • site-directed mutagenesis or randomly generated mutations within the EcR or FXR may be recovered.
  • Such mutations may include sequences that enhance the induced responsiveness of the cell culture system, and/or reduce non-induced transcription levels while maintaining or increasing induced transcriptional activity, and/or change the specificity or induction properties of the receptor for the purposies of screening a subclass or potential insecticide compounds.
  • the assay may use DNA constructs isolated from various species to develop species-specific insecticides.
  • DNA constructs isolated from various species may have different activities.
  • the FXR or EcR constructs and/or their respective heterologous binding partners may comprise species-specific constructs.
  • the DNA encoding the ecdysone receptor (EcR) or the farnesoid X-activated receptor (FXR) may comprise a chimera of DNA from different insect or vertebrate sources.
  • the chimera may comprise a mammalian activating domain.
  • the chimera may comprise a mammalian DNA binding domain (DBD) and/or a mammalian ligand binding domain (LBD).
  • the mammalian receptor domains e.g., activating, DBD, or LBD
  • the chimera may comprise an EcR-activating domain.
  • the chimera comprises an EcR DNA binding domain (DBD) and/or an EcR ligand binding domain (LBD).
  • the EcR sequences may be derived from an arthropod.
  • the EcR sequences may be derived from Drosophila melanogaster .
  • the chimera may comprise a viral protein-16 (VP16) activating domain from pseudorabies virus.
  • the chimera may comprise Chironomus tentans (lower Diptera) (Cf) USP LBD, such as utilized in the VP16CfUSP construct.
  • portions of the activating domain, DNA-binding domain, and hinge region may be deleted from either the natural EcR or USP receptor.
  • one of the three characterized mammalian (e.g., mouse, rat, or human) RXRs may be used as the heterologous binding partner.
  • LBDs Manduca Sexta (Lepidoptera), Locusta migratoria (Orthoptera), Heliothis virescens (Leptidoptera), Apis mellifera (Hymenoptera), Aedes aegypti (lower Diptera), and Tenebrio molitor (Coleoptera) may also be employed.
  • the present invention also comprises compounds that act as insecticides by their ability to inhibit insect growth. Also, the present invention may comprise a use for compounds identified using the methods and systems of the invention as insecticides and/or modulators of insect growth.
  • the present invention comprises a composition for use as an insecticide comprising a compound that activates FXR-mediated transcription or EcR-mediated transcription mixed with a suitable carrier for application to plants.
  • an increase of EcR-mediated transcription by the compound may comprise potentiation of hormone induced EcR transcription.
  • the hormone comprises an ecdysteroid.
  • the hormone may comprise murA or 20-hydroxyecdysone (20E).
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise farnesol or a farnesol metabolite.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise a juvenile hormone mimetic.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise a plant-derived JH agonist.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise an insecticide synergist.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may also comprise a monoterpene.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription comprises a diterpene.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise a triterpene.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may also comprise a furocoumarin.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may also comprise a phenylpropanoid.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription comprises a coumarin.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise a flavonoid.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprises a linoleic acid metabolite.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise a polyketide.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription comprises a xanthine.
  • compounds that increase FXR-mediated transcription or EcR-mediated transcription may comprise man-made compounds.
  • the compound that increases FXR-mediated transcription or EcR-mediated transcription may comprise an organochlorine.
  • a “ligand” is a molecule that interact either directly or indirectly with a receptor to form a complex.
  • An “agonist” comprises a compound that binds to a receptor to form a complex that elicits a pharmacological response specific to the receptor involved.
  • an “antagonist” comprises a compound that binds to an agonist or a receptor to form a complex that does not give rise to a substantial pharmacological response and can inhibit the biological response induced by an agonist.
  • Polypeptide and “protein” are used interchangeably herein to describe protein molecules that may comprise either partial or full-length proteins.
  • a “polypeptide domain” comprises a region along a polypeptide that comprises an independent unit. Domains may be defined in terms of structure, sequence and/or biological activity. In one embodiment, a polypeptide domain may comprise a region of a protein that folds in a manner that is substantially independent from the rest of the protein. Domains may be identified using domain databases such as, but not limited to PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.
  • nucleic acid is a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term is used to include single-stranded nucleic acids, double-stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogues.
  • vector refers to a nucleic acid molecule that may be used to transport a second nucleic acid molecule into a cell.
  • the vector allows for replication of DNA sequences inserted into the vector.
  • the vector may comprise a promoter to enhance expression of the nucleic acid molecule in at least some host cells.
  • Vectors may replicate autonomously (extrachromasomal) or may be integrated into a host cell chromosome.
  • the vector may comprise an expression vector capable of producing a protein derived from at least part of a nucleic acid sequence inserted into the vector.
  • fusion protein may refer to a protein or polypeptide that has an amino acid sequence derived from two or more proteins.
  • the fusion protein may also include linking regions of amino acids between amino acids portions derived from separate proteins. Unless specifically stated, there is no required order of linking polypeptides to form a fusion protein.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning two sequences and refers to the number of identical residues (i.e., amino acid or nucleotide) at positions shared by the compared sequences. Sequence alignment and comparison may be conducted using the algorithms standard in the art (e.g. Smith and Waterman, Adv. Appl. Math., 1981, 2:482; Needleman and Wunsch, 1970 , J. Mol. Biol., 48:443); Pearson and Lipman, 1988 , Proc. Natl. Acad. Sci.
  • percent identity of two sequences may be determined using GCG with a gap weight of 1, such that each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • an “effective amount” as used herein means the amount of an agent that is effective for producing a desired effect. Where the agent is being used to achieve a insecticidal effect, the actual dose which comprises the effective amount may depend upon the route of administration, and the formulation being used.
  • modulation of insect growth includes the modulation of the growth of an individual insect or an insect population and includes modulation of insect reproduction, morphogenesis, and survival.
  • insect growth comprises growth and development of an individual insect and/or an insect population and thus, refers to the growth, morphogenesis, and survival of an individual insect, or an insect population.
  • an “increase in transcription” comprises an increase from a measurable basal level to a higher level.
  • an “increase in transcription” may comprise an increase from an undetectable level to a measurable level.
  • activation of FXR or “activation of EcR” describes increasing FXR-mediated transcription or EcR-mediated transcription, respectively.
  • FXR-mediated transcription comprises a gene transcription event that requires binding of the activated farnesoid-X-receptor to a promoter upstream of the gene being transcribed.
  • EcR-mediated transcription comprises a gene transcription event that requires binding of the activated ecdysone receptor to a promoter upstream of the gene being transcribed.
  • hormone activated transcription comprises an increase in transcription induced by hormones that bind to hormone response elements upstream of the gene being transcribed.
  • a “hormone response element” comprises a nucleotide region upstream of a gene that mediates the effect of a steroid hormone.
  • an “isoform” is a variant form of a protein that has the same general function as another protein but which may have small differences in its sequence either because it is encoded by a different gene, is expressed by a different promoter in the same gene, or is derived by alternative splicing of the same pre-mRNA.
  • EcR may exists in at least three versions having trans-activating regions that differ in sequence to provide isoforms EcRA, EcRB, and EcRB2, each of which can activate transcription of a gene having an EcR HRE in its promoter.
  • EcRA is derived from a different promoter of the EcR gene, and B1 and B2 are derived from alternative splicing of a pre-mRNA.
  • a “ligand binding domain” is that portion of a protein or polypeptide involved in binding of a ligand.
  • a “juvenile hormone mimetic” is a compound that functions like any one of the natural juvenile hormones such as JHI, JH II, or JH III.
  • the normal physiological functions of the naturally-occurring compounds are compromised by ectopic or exogenous administration of any number of juvenile hormone mimetic compounds.
  • insecticide synergist is a compound that acts in synergy with an insecticide to provide a response that is greater than additive.
  • a “monoterpene” is an acyclic or cyclic C 10 hydrocarbon composed of two isoprene units and their oxygenated derivatives. Common monoterpenes include geraniol, limonene, ⁇ -pinene, camphor.
  • a “sesquiterpene” is an acyclic or cyclic C 15 hydrocarbon and their oxidized derivatives composed of three isoprene units.
  • a “diterpene” is a acyclic or cyclic C 20 hydrocarbon and their oxidized derivatives composed of four isoprene units.
  • a “triterpene” is a acyclic or cyclic C 30 hydrocarbon and their oxidized derivatives composed of six isoprene units.
  • coumarin is 2H-1-Benzopyran-2-one.
  • a “furocoumarin” is a psoralen derivative of coumarin such as bergamotin.
  • phenylpropanoid is a compound derived from phenylalanine and cinnamic acid.
  • a “flavonoid” is a phenolic compound build up of two aromatic rings and held together by a C3 unite. Flavonoids include chalcones, isoflavanoids (rotenone), flavolignans (silybin).
  • a “polyketide” included molecules synthesized form acetyl CoA, propionyl CoA, butyryl CoA, malonyl CoA, methylmalonyl CoA, and ehtylmalonyl CaA intermediates.
  • the present invention recognizes that a variety of natural and synthetic insecticides can act to modulate transcriptional activity programmed by FXR. These putative insecticides may also potentiate Drosophila EcR-mediated transcription in the presence of limiting amounts of an ecdysone, thereby providing an assay system for the development of new insecticides.
  • the present invention describes compounds that activate FXR and/or EcR as potential insecticides.
  • the present invention also describes the use of such FXR-activating and EcR-activating and/or potentiating compounds isolated from plants or plant metabolites as environmentally friendly insecticides.
  • JH may act upon transcriptional activity, and may modulate the transcriptional regulation of ecdysteroids (Cherbas, L., et al., 1989 , Dev. Genet., 10:177-188; Farkas, R., and J. Knopp, 1997 , Arch. Insect Biochem. Physiol., 35, 71-83; Hiruma, K., et al., 1999 , Dev. Genes Evol., 209, 18-30; Zhou, X., and L. M. Riddiford, 2002 , Development, 1290: 2259-2269).
  • Ultraspiracle is a nuclear receptor that may dimerize with the ecdysone receptor (EcR) to form the functional ecdysteroid receptor complex (Thomas, H. E., et al., 1993 , Nature, 362:471-475; Yao, T. P, et al., 1993 , Nature, 366:476-479; Riddiford, L. M, et al., 2000, Vitam. Horm., 60: 1-73). USP is an insect orthologue of the vertebrate retinoid X receptor (RXR) (Oro, A.
  • FXR farnesoid-activated X receptor
  • FXR induces transcription from a promoter containing the heat shock protein 27 (hsp27) ecdysone response element (EcRE) (Riddihough, G., and H. R. Pelham, 1987 , EMBO J., 6:3729-3734).
  • hsp27 heat shock protein 27
  • EcRE ecdysone response element
  • LBD ligand-binding domain
  • the present invention recognizes that there may be overlap between the mammalian FXR/RXR system and the insect EcR/USP systems, and provides numerous plant-derived and man-made JHs that have the ability to increase transcriptional activity programmed by FXR and/or EcR.
  • the present invention comprises assay systems and methods to test various EcR isoforms and chimeras as having the ability mediate a transcriptional response to ecdysteroids and JHs.
  • the ability to reconstitute ecdysteroid responsive transcriptional effects in an otherwise non-responsive mammalian cell culture (Christopherson, K. S., et al., 1992 , Proc. Natl. Acad. Sci.
  • the present invention comprises methods and systems to evaluate the ability of specific compounds to activate either FXR or EcR mediated transcription, wherein compounds exhibiting the ability to activate EcR or FXR mediated transcription comprise potential modulators of insect growth and/or insecticides.
  • Example embodiments of assay systems of the present invention are depicted schematically in FIGS. 1A and 1B .
  • the assay systems as depicted in FIGS. 1A and 1B are non-limiting in that the mechanism of interaction of the various assay components may vary from that depicted in the drawing, while still providing a functional assay system.
  • the compound to be tested, (X) may bind directly to FXR or EcR protein as indicated schematically in FIG. 1 .
  • compound (X) may modify FXR (or EcR) activity without directly binding to the protein, but indirectly in some manner.
  • the assay system may comprise four components.
  • the first component may comprise a DNA construct that encodes a functional EcR.
  • a cell shown as the large oval in FIG. 1
  • the EcR polypeptide may comprise an EcR chimera comprising a mammalian (e.g., human) glucocorticoid receptor (GR or hGR) trans-activation domain (A/B) attached to an insect (e.g., Drosophila ) EcR DNA binding domain and hinge (DBD) (C/D) and ligand binding domain (LBD) (E).
  • GR or hGR glucocorticoid receptor
  • the construct may contain a region (F) that may function as a transactivation domain (Palli, S. R. et al., 2003 , Eur. J. Biochem., 270:1308-1315)
  • the second component of the assay may comprise the binding partner needed for EcR (or FXR) activity.
  • a cell may therefore be co-transfected with a second expression plasmid comprising sequences that encode a mammalian RXR (e.g., mouse, rat, or human RXR ⁇ ) or an insect USP.
  • These proteins may also comprise a trans-activating domain (A/B) and a DNA binding domain (C) and hinge (D), and a ligand binding domain (E).
  • RXR and/or USP may not necessarily comprise an (F) domain.
  • the plasmids may be co-transfected into a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, so that there is no endogenous EcR or ecdysteroid present.
  • the third component of the assay system may be an exogenous ecdysteroid such as muristerone A (murA), that may act to induce EcR dependent transcription.
  • the assay system may comprise a means to measure EcR- or FXR-mediated transcription.
  • the response of transfected cells to a compound (X) that is able to increase FXR-mediated transcription or EcR-mediated transcription may be measured using a reporter plasmid bearing a EcR (or FXR) hormone responsive element (HRE).
  • the reporter plasmid may comprise a construct having multiple copies of the hormone-responsive element inserted upstream of a gene having a measurable gene product.
  • multiple copies of an ecdysone-responsive element, hsp27 EcRE are inserted into the mouse mammary tumor virus (MTV) promoter positioned upstream of the bacterial chloramphenical acetyltransferase gene to generate an (EcRE) 5 - ⁇ MTV-CAT reporter plasmid.
  • MTV mouse mammary tumor virus
  • the reporter gene may comprise the luciferase gene (LUC) or a green fluorescent protein (GFP).
  • the reporter plasmid comprises an (EcRE) 5 - ⁇ MTV-LUC construct produced as described herein. Using the luciferase gene may allow for FXR or EcR activated transcription to be measured in-situ by monitoring the luminescence of the cells or organism being used in the assay.
  • FIG. 1B shows a non-limiting example embodiment of an assay system for FXR-mediated transcription and measurement of the ability of a compound, (X), to modulate FXR-mediated transcription.
  • a hormone such as murA
  • compound (X) may modify transcription of the reporter gene that is mediated by FXR, but that does not require or employ murA.
  • the compound to be tested, (X) may bind directly to FXR or EcR protein, or it may modify FXR (or EcR) activity without directly binding to the protein, but indirectly in some manner.
  • cells may be transfected with plasmids comprising a GRdEcR chimera that consists of the rat glucocorticoid receptor (GR) activation domain (A/B) attached to the Drosophila melanogaster (d or Dm) EcR DNA binding domain (DBD) (C/D) and ligand binding domain (LBD) (E), and a second plasmid comprising mouse RXR (mRXR ⁇ ).
  • GRdEcR chimera that consists of the rat glucocorticoid receptor (GR) activation domain (A/B) attached to the Drosophila melanogaster (d or Dm) EcR DNA binding domain (DBD) (C/D) and ligand binding domain (LBD) (E)
  • a second plasmid comprising mouse RXR (mRXR ⁇ ).
  • the response of transfected cells to the ecdysteroid, murA may be measured using a (EcRE) 5 - ⁇ MTV-CAT reporter plasmid that carries five tandem repeats of the hsp27 EcRE linked to the MTV promoter and the chloramphenicol acetyltransferase (CAT) gene.
  • EcRE europentase
  • CAT chloramphenicol acetyltransferase
  • hormone e.g., muristerone A
  • the dose of hormone may range from 0.001 ⁇ M to 5.0 ⁇ M, or from 0.005 ⁇ M to 0.5 ⁇ M, or from 0.01 ⁇ M to 0.1 ⁇ M.
  • the transfected cells comprise mammalian cells.
  • mammalian cells By using mammalian cells as the host, cofactors and transcription factors are present, but there may be minimal background activity due to endogenous ecdysone receptor and/or ligands. Any cell type that does not normally respond to ecdysteroids, but having the required transcription factors is a potentially suitable cell type for the assay.
  • the present invention comprises the use of specific receptor isotypes to evaluate potential insecticides.
  • the assay may use an arthropod (e.g., insect) ecdysone receptor (EcR), an insect USP, and a suitable reporter gene construct (e.g., CAT or luciferase linked to a HRE specific to FXR or EcR).
  • the assay may comprise using an insect EcR with a mammalian RXR, and a suitable reporter gene construct.
  • the assay may comprise use of an FXR construct in combination with a mammalian RXR construct.
  • the assay may comprise using FXR in combination with USP.
  • Nuclear receptors such as EcR and FXR, may comprise three general domains structural domains: (1) a transcriptional activating domain—A/B; (2) a DNA binding (DBD)—C; and (3) a hinge (D) and ligand binding domain (LBD) (E) ( FIG. 1 ).
  • the hinge (D) may be considered as part of the DBD (C) or the LBD (E) when describing various nuclear receptor protein constructs.
  • a C-terminal (F) domain may comprise trans-activation characteristics.
  • the EcR construct may comprise the human glucocorticoid receptor (GC) or (hGC) activating domain (e.g., A/B) and the Drosophila (d or Dm) EcR DNA binding domain (C) and the Drosophila hinge-ligand binding domain (D/E).
  • a VP16EcR construct comprising the VP16 activating domain (VP16) in combination with Drosophila EcR DBD and LBD domains (EcR) may be used.
  • the assay may also comprise a construct, GGEc, comprising the human glucocorticoid receptor (G) trans-activating domain and DBD with the Drosophila EcR (Ec) LBD.
  • the assay may also comprise a construct, GEcEc, comprising the human glucocorticoid receptor (G) activating domain and the Drosophila (Ec) DBD and LBD.
  • the assay may comprise a construct, GGF, comprising the human glucocorticoid receptor (G) activating domain and DBD with the FXR (F) LBD derived from vertebrate species (avian, rodent, human).
  • the assay comprises a construct, VP16CfUSP, comprising the VP16 activation domain directly linked to the LBD (i.e., D/E domains) of Choristoneura fumerifana (Cf) (spruce budworm) USP.
  • the VP16 trans-activating domain may be linked to the Drosophila USP protein DBD and LBD to generate the contruct, VP16-DmUSPDBD-DmUSPLBD.
  • natural isoforms of Dm EcR with A/B domains A, B1 or B2 with the F domain deleted may be used.
  • Table 2 provides several non-limiting constructs that may be used with the methods and systems of the present invention, utilizing nomenclature that is standard in the art.
  • RXR and USP expression plasmids may be used in the assay of the present invention.
  • expression plasmids may have promoter elements and polyadenylation signals between which nucleic acid sequences encoding the amino acids of interest are positioned.
  • the plasmids produce RXR or USP proteins in the presence of the appropriate transcription factors.
  • Promoter elements for the RXR and USP proteins may be such as to drive transcription in a constitutive fashion. These typically include those from Rous sarcoma virus or cytomegalovirus.
  • chimeric DNA constructs allows for the construction of nuclear receptors which interact with mammalian transcriptional factors to induce or repress transcription mediated by the DBD and/or LBD of the insect receptor, EcR, and its partner, USP. Because the mammalian cells do not contain endogenous EcR or USP, there is little or no endogenous response to the experimental ligand.
  • the A/B domain of EcR may be responsible for modifying transcriptional activity of the receptor.
  • various isoforms of the EcR A/B domain e.g., A, B1, or B2
  • the system may display specificity between a particular EcR isoform and the heterodimeric partner or the ability of the EcR to respond to the test agent (X).
  • different EcR/USP and EcR/RXR heterodimeric combinations display different responsiveness to the test agent (X).
  • juvenile hormones can potentiate the effect of EcR ligands.
  • a GRdEcR chimera that consists of the human glucocorticoid receptor activation domain (GR) attached to the Drosophila melanogaster (d) EcR DBD and LBD was cotransfected with mouse RXR (mRXR) into CHO cells.
  • GR human glucocorticoid receptor activation domain
  • JHIII juvenile hormone III
  • EcRE embryonic hormone III
  • JHIII may potentiate the response of murA in a dose-dependent manner (using 20, 40, 80, and 160 ⁇ M JHIII) at submaximal ecdysteroid dosages (0.1 ⁇ M and 1 ⁇ M murA) ( FIG. 2 , sets 2 and 3, respectively).
  • JHs can potentiate the effects of ecdysteroid ligands
  • JHIII may not increase the response above the maximal response seen with high concentrations of the ecdysteroid.
  • JHIII does not evoke a response that is greater than the maximal level induced by 10 ⁇ M murA.
  • a mammalian cell-based assay system allows for controlled addition of the components required for activity.
  • juvenile hormones do not interact with unbound EcR, but require addition of submaximal levels of exogenous hormone for activity.
  • JHIII alone shows no effect on transcription mediated by the GRdEcR chimera with RXR ( FIG. 2 , set 1).
  • Ecdysteroid responsiveness and JHIII potentiation in EcR chimeras may depend, at least in part, upon the activating ligand being used.
  • Ecdysteroid responsiveness and JHIII potentiation in EcR chimeras may also depend upon and the heterodimeric partner used in the assay system.
  • transfection of mammalian CHO cells with the VP16 activation domain linked to the DBD and LBD of Drosophila melanogaster (Dm or d) EcR may result in a sensitive and robust ecdysteroid response to muristerone A.
  • FIG. 3 shows the ecdysteroid response and potentiation effects of JHIII measured for various EcR combinations and mouse RXR in accordance with an embodiment of the present invention.
  • mouse RXR was used as the heterologous dimer and a luciferase construct, (EcRE) 5 - ⁇ MTV-LUC, was used as the reporter gene. Shown in FIG.
  • VP16dEcR/mRXR at 0.01 ⁇ M murA 3 a
  • VP16dEcR/mRXR at 0.1 uM murA 3 b
  • VP16dEcR/VP16CfUSP at 0.1 ⁇ M murA
  • EcRA/mRXR at 1 ⁇ M murA 3 d
  • EcRB1/mRXR at 0.1 ⁇ M 3 e
  • EcRB2/mRXR at 0.1 ⁇ M murA ( 3 f ), each in the presence, or absence, of 80 ⁇ M JHIII.
  • VP16dEcR viral transactivation A/B domain linked to Drosophila (EcR) LBD and DBD
  • mouse RXR may respond to 0.01 ⁇ M and 0.1 ⁇ M murA ( FIGS. 3 a and 3 b ).
  • the response of the VP16dEcR/mRXR system is generally strong and robust as compared to other combinations of binding and transcriptional activation units.
  • the response of the VP16dEcR/mRXR system may be further potentiated by JHIII.
  • the VP16dEcR chimera may also display a discernible response to 20E at 10 ⁇ M (e.g., over 20-fold) using RXR.
  • VP16dEcR/20E activity is not, however, necessarily potentiated by JHIII.
  • VP16dEcR/RXR in the presence of 20E is only minimally affected by the additional presence of JHIII (not shown).
  • the assay of the present invention may allow for the mixing of different molecular constructs as a means to better understand the molecular nature of insect development and insecticide structure and function.
  • the VP16dEcR construct may also be tested with VP16CfUSP (Table 2) ( FIG. 3 c ). Using this system, the same degree of potentiation may occur for murA using the VP16EcR/USP combination as with the VP16dEcR/RXR complex, except that a higher murA dose may be required to achieve the same efficacy.
  • the assay of the present invention provides for the use of different ecdysone receptor isoforms, either natural or generated by mutagenesis, as a means to evaluate the activity of various test compounds.
  • the assay may comprise the use of various FXR isoforms, either natural or generated by mutagenesis (Zhang, Y, et al., 2003 , J. Biol. Chem. 278: 104-110).
  • EcR isoforms such as the three natural Drosophila melanogaster EcR isoforms, EcRA, EcRB1, and EcRB2 may be cotransfected into CHO cells with either USP or RXR expression plasmids such as, but not limited to, the VP16CfUSP and/or VP16CRXR fusion proteins.
  • the different EcR isoforms e.g., Drosophila A, B1, and B2
  • the isoforms may display unique expression profiles among each other and as compared to VP16dEcR/RXR (not shown).
  • the activity observed among the three EcR isoforms may depend, at least in part, upon the identity of the heterologous partner used.
  • the EcRB1/VP16CfUSP combination shows a relatively high level of ligand-independent transcription, with between 10 and 20-fold higher basal levels than other EcR constructs.
  • the EcRA isoform in combination with VP16CfUSP may also show a basal level of transcription that is 2 to 3 fold higher than the basal level of transcription obtained with EcRB2 isoform.
  • the basal activity of the EcRB2/VP16CfUSP dimer may be about the same as the basal activities produced by VP16dEcR/RXR and GREcR/RXR (not shown).
  • all three Drosophila isoforms may be induced by about 30-40 fold at 1 ⁇ M murA.
  • the response of all EcR isoforms in the presence of VP16CfUSP is potentiated by the presence of 80 ⁇ M JHIII in the presence of 0.1 ⁇ M murA.
  • Data showing induction of isoforms A (set 1), B1 (set 2) and B2 (set 3) by murA, and potentiation by JHIII is shown in FIG. 4 .
  • the JH potentiation is dose-dependent, similar to the results seen with GrdEcR.
  • EcR N-terminal domain and the heterodimeric partner may therefore result in a functional receptor that is capable of showing an ecdysteroid response and/or JHIII potentiation.
  • the potentiation by JHs is not due to the activation of RXR by either JHIII or a JHIII metabolite, as JHIII by itself shows no potentiation of transcriptional activity.
  • the differential ecdysteroid- and JHIII-dependent transcriptional activities noted among the EcR isoforms may offer insights concerning the lack of correspondence between cellular isoform titers and developmental effects in Drosophila tissues.
  • the B isoforms may be functionally distinguished from the EcRA isoform (Bender, M., et al., Genetics 91:777-788), and recent studies have distinguished biological roles for B1 and B2 (Cherbas, L., et al., 2003 , Development 130:271-284).
  • the B2 N-terminal domain is shorter than the B1 N-terminal domain, and is capped with an amphipathic helix (Talbot, W.
  • combinations of EcR/USP, EcR/RXR, and FXR/RXR show a response to murA that may be potentiated by JH ( FIG. 5 ).
  • the combination of FXR with USP may not respond to JHIII.
  • the FXR activator JHIII can potentiate the transcriptional response of EcR induced by ecdysteroids, it appears that in at least some embodiments, the JHIII response exhibited by FXR may require RXR, and not USP, as a heterodimeric partner.
  • USP and RXR may not be interchangeable.
  • EcR may be a prerequisite for observing the potentiative effects of JHIII on EcR.
  • Assays using the various EcR forms indicate that multiple factors may influence receptor activity, including the activating ecdysteroid, the N-terminal domain of EcR, and the heterodimeric partner.
  • the combination of FXR and USP may evoke a low level response in CHO cells to JHIII ( FIG. 5 ) attributable to endogenous expression of low levels of RXR in these cells, where the addition of ecdysteroids (murA or 20E) with JHIII induces no further elevation of FXR/USP-mediated activity ( FIG. 5 ).
  • EcR and USP may be unable to potentiate a response to 20 ⁇ M chenodeoxycholic acid (CDCA), the most efficacious naturally-occurring activator of FXR known to date.
  • the three isoforms may respond to 20E only in conjunction with their natural partner, USP.
  • 20E responsiveness may involve a compatibility between the N-terminal domain of EcR and the heterodimeric partner.
  • the high level of B1 basal activity may relate to a cell-specific aspect of CHO cultures, as this effect has not been seen in HeLa cells.
  • the selectivity of the 20E responsiveness may be further substantiated by the ability of the VP16dEcR/VP16CfUSP combination to respond only to murA, but not to 20E.
  • + designates a change in transcriptional level that exceeds 2.5-fold
  • ++ indicates a response at a lower dosage than other EcR forms.
  • Responses are based on dosages of 0.1 ⁇ M murA, 10 ⁇ M 20E, and 80 ⁇ M JHIII.
  • (+) indicates inducibility; for columns involving JHIII, (+) indicates observed potentiation that exceeds 2-fold above the levels observed with ecdysteroid alone.
  • the activating ecdysteroid may also determine the ability of JHIII to potentiate a response in other EcR dimers.
  • the VP16dEcR/RXR combination is responsive to both 20E and murA; but when 20E is used, JHIII may not be capable of potentiating the response, whereas JHIII strongly potentiates the murA response.
  • EcR bound to its cognate ligand, may acquire a conformation that allows further activation by JHIII either directly or via an indirect interaction.
  • the amount of ligand-bound EcR may be limiting factor in the cell-based assay of the present invention. For example, while the amount of potentiation JHIII potentiation may remain constant over a range of submaximal murA doses, the absolute transcriptional activity attributable to a fixed JHIII dose may increase as ecdysteroid molarity increases, indicating that the number of ligand-activated EcR proteins may be a rate-limiting factor.
  • RXR may be activated through its LBD by methoprene acid, a metabolite of methoprene, and with the retinoic acid receptor (RAR) generates a response to the ligand through a direct repeat element (Harmon et al., 1995).
  • RXR ligands may also increase the responsiveness of VP16dEcR to murA via the hsp27 response element (Saez, E., et al., 2000 , Proc. Natl. Acad. Sci. USA, 97:14512-14517) to supra-maximal levels.
  • FXR Farnesoid X-activated Receptor
  • EcR Ecdysone Receptor
  • Farnesol was the first molecule to be recognized as a juvenoid (P Schmialek, 1961 , Z. Naturf., 16b: 461-464).
  • JH juvenile hormone
  • JH III the most widely distributed juvenile hormone (JH) in the arthropod world, JH III, was synthesized via bioassay-guided structural optimization based upon farnesol (Bowers, W. S., et al., 1965 , Life Sci., 4:2323-2331) prior to its identification from insect extracts (Roller, H., et al., 1967 , Angew. Chem. Int. Ed., 6:179-180).
  • rational design has provided a platform for the development of numerous drugs and vaccines important for human and veterinary health, and provides a conceptual basis for the future design of more effective insecticides.
  • biologically important molecules are characterized by comparing structure with activity in order to identify functional groups that either enhance, alter, or block activity.
  • Farnesol is metabolized from farnesyl diphosphate, a precursor to cholesterol, ubiquinones, dolichols, and other growth-requiring isoprenoids (Goldstein, J. L., and M. S. Brown, 1990 , Nature 1990, 343:425-430).
  • compounds derived from everyday plants such as farnesol and other juvenoids, and including those that may constitute a portion of the human diet, may comprise the ability to activate FXR and/or EcR (i.e., to increase FXR-mediated transcription or EcR-mediated transcription, repectively) and thus, may function as insecticides and/or insect growth regulators.
  • the compounds that increase FXR and/or increase or potentiate EcR-mediated transcription comprise farnesol metabolites.
  • farnesyl diphosphate is metabolized to juvenile hormone III ( FIG. 6 ), and at least some of the intermediate metabolites may interact with FXR to induce RXR mediated transcription.
  • farnesol, nerolidol, and JHIII may induce FXR greater than 10-fold.
  • farnesal, farnesoic acid, and methyl farnesoate also interact with FXR to induce RXR mediated transcription.
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription may comprise juvenile hormone mimetics.
  • FXR-activating famesoids have been described as JH agonists in insect bioassays (Schneiderman, H. A., and L. I. Gilbert, 1964 , Science, 1964, 143:325-333).
  • the JH mimetics comprise farnesol, neorlidol, and phytol (Table 4), as well as the synthetic juvenoids methoprene, pyriproxyfen, and the ethyl ester of 7,11-dicholoro-2-ene famesoic acid ( FIG. 7A ).
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription comprise plant-derived JH agonists.
  • the plant derived JH agonists that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise echinacea oil, echinolone, juvocimene, juvabione, ⁇ -bisabolol, olive oil, 2-hydroxyphenethlyl alcohol, 3-hydroxyphenethlyl alcohol, or 4-hydroxyphenethlyl alcohol ( FIG. 7B ).
  • the compounds that increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription comprise insecticide synergists.
  • the insecticide synergists that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise piperonyl butoxide (PB), seasamin, sesame oil, piperine, myristicin, or apiole ( FIG. 7C ).
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription comprise monoterpenes.
  • the monoterpenes that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise tea tree oil (terpenen-4-ol, 1,8-cineole, and ⁇ -terpineol), carvacrol, thymol, perillyl alcohol, fenchyl alcohol, or pinane diol (Table 4 and FIGS. 8A and 8B ).
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription may also comprise diterpenes.
  • the diterpenes that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise forskolin, 1-trans- ⁇ 9 -tetrahydrocannabinol (THC), abietic acid, croton oil, and other phorbol-like diterpenes such as phorbol 12,13-dibutyrate, mezerein, and also ingenol 3,20-dibenzoate, cafestol, kahweol, or their acetate derivatives.
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription may comprise triterpenes.
  • the triterpenes that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise essential oils from myrrh and frankincense, ⁇ -boswellic acid, oleanolic acid, rosemary oil, or 20 ⁇ - or 20R-hydroxycholesterol.
  • the present invention also comprises compound that can suppress the activity of FXR or EcR.
  • Compounds that inhibit the ecdysone receptor (EcR) may suppress FXR activity.
  • the compounds that inhibit FXR activity may suppress FXR activity promoted by FXR modulators.
  • 1 ⁇ M cucurbitacin D (cuc D) may suppress FXR-dependent activity promoted by JH III and chenodeoxycholate (CDCA) (40 ⁇ M each), 7-fold and 58-fold, respectively ( FIG. 8C ).
  • the ⁇ 1 -unsaturated congener cucurbitacin I may inhibit farnesol-induced activity with an IC 50 ⁇ 50 nM (data not shown).
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription may also comprise furocoumarins or phenylpropanoids.
  • the fucocoumarins and phenylpropanoids that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise the furocoumarins, bergamot oil and bergamotin (from Earl Grey tea) ( FIG. 8D ), myristicin, or apiole, or the phenylpropanoid, methyleugenol ( FIG. 8E ).
  • the compounds that increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription may comprise coumarins and flavanoids.
  • the coumarins and flavanoids that increase FXR-dependent transcription may, for example, comprise silybin, tangeretin or a rotenonic acid ( FIG. 8F ) or 8-prenylnaringenin and isozantholhumol from hops.
  • the compounds that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription may also comprise linoleic acid metabolites.
  • the linoleic acid metabolites that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise cis-jasmone or methyl jasmonate.
  • the compounds that increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription comprise polyketides from hops.
  • the polyketides that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise humulone ( FIG. 8G ).
  • the compounds that increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription comprise xanthines.
  • the xanthines that increase FXR-dependent transcription, and/or increase or potentiate EcR-mediated transcription comprise theophylline, caffeine, 8-Br-cAMP, dibutyryl cAMP, or 8-Br-cAMP in combination with theophylline ( FIG. 8H ).
  • man-made insecticides may increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription.
  • man-made insecticides that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription comprise cinerins, pyrethrins, jasmolins, synthetic pyrethroids including cypermethrin, permethrin, phenothrin, and bioallethrin (Table 5).
  • jasmolin N.T. rice N.T. ⁇ -tocotrienol 20 1 sage 8 sesame 3 sesamin 100 12 sesamol 1 spruce N.T. abietic acid 50 23 tea tree 8 1,8-cineole 400 2 ⁇ -terpineol 402 terpinen-4-ol 400 2 thyme 13 vetiver 24 ylang ylang 17 cis-jasmone 1000 6 methyl jasmonate 1000 18
  • FXR activity is defined as the ratio of FXR-dependent CAT activity attained in appropriately transfected cells at maximal doses of plant oil (or indicated doses of ingredients) over that for the vehicle. Activity level of “1” indicates no increase in activity over vehicle.
  • insecticides such as o,p-DDT (but not p,p-DDT), chlordane, kepone, lindane, dieldrin, toxaphenes, aroclor 1254, 2,3,7,8-tetrachlorodibenzo- p -dioxin, malathion, diazinon, chlorpyrifos, parathion, ethion, chlorfenapyr, pyrethrin, permethrin, fenvalerate, or fipronil may increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription in an embodiment of the present invention (Table 6; FIG. 9 ).
  • the present invention recognizes that small doses of ecdysone may prime the EcR-dependent transcriptional response provoked by JHs.
  • Ecdysones may stabilize a conformation of EcR that permits JHs to bind more effectively. Since JH potentiators may affect the maximal transcriptional response elicited by ecdysone, this may be similar to allosteric enzymes whose effectors alter the apparent V max without changing the K m value.
  • the variable cytotoxicities provoked by dietary or ectopically-applied ecdysones, JHs, or insecticides in different insects may be uniquely imparted by their pharmacologically-distinguishable EcR/USP complexes.
  • the present invention describes the use of JH antagonists as compound that may be used to modulate insect growth.
  • the JH antagonists may increase FXR-mediated transcription.
  • the JH antagonists may inhibit FXR-mediated transcription.
  • Precocene I can be inactive in RAR, RXR, PPAR, or GR-based transcriptional assays, which suggests that it may be relatively specific for FXR (data not shown).
  • FXR may be activated by other precocene-like JH antagonists ( FIG. 10C ) including 3,4-dimethoxy-6-isopentenylphenol (3-fold induction at 100 ⁇ M) and a tricyclic dichromene (29-fold induction at 25 ⁇ M), di-, tri-, and tetraprenylated ubiquinones (U2, U3, and U4) ( FIG. 10F ).
  • FXR may be inhibited by precocene metabolites and derivatives, such as 6,7-dihydroxy precocene and 6,7-dihydroxy coumarin (esculetin), and ubiquinone-1 ( 10 B, 10 C, and 10 D).
  • precocene metabolites and derivatives such as 6,7-dihydroxy precocene and 6,7-dihydroxy coumarin (esculetin), and ubiquinone-1 ( 10 B, 10 C, and 10 D).
  • transcriptional activity programmed by muristerone-primed ecdysone receptors may be potentiated by juvenile hormones, compounds derived from food sources, and insecticides ( FIG. 11 ).
  • the transcriptional effects may, in some embodiments, require ligand binding domain sequences of the EcR or FXR ( FIG. 12 ).
  • mutations in the LBD of FXR or EcR may be utilized to prepare constructs specific to various types of potential insecticide compounds.
  • natural JH's may increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription.
  • FXR may respond to the JH I analog ZR354 in which the ethyl groups of JH I ethyl are replaced by similarly bulky dimethyl groups.
  • the natural JH's that increase FXR-mediated transcription, and/or increase or potentiate EcR-mediated transcription comprise all “natural” JHs, e.g., JH 0, JH1, and JH II (where ethyl groups are substituted for JH III methyl groups) ( FIG. 13 ).
  • Compounds of the present invention may be used in the form of compositions and can be applied to the crop and/or plant to be treated, simultaneously with, or in succession with, other compounds such as fertilizers, micronutrient donors or other preparations which influence the growth of plants.
  • the compounds can also be selectively combined with herbicides, as well as, other insecticides, fungicides, bactericides, nematocides, molluscicides or mixtures of several of these preparations and, if desired together with further carriers, surfactants or application promoting adjuvants employed in the art of formulation, and as described in U.S. Pat. Nos. 6,737,383, 6,630,465, 6,586,470, 6,603,044, 6,617,341, 5,942,542, and 5,849,320.
  • the compound of the present invention when applying the compound of the present invention, the compound may be applied in a form as it is without adding other active components.
  • the compound of the present invention when applied for plant protection purpose, the compound can be prepared into general types of formulations for plant protection use, such as wettable powder, granules, dust, emulsifiable concentrate, water soluble powder, suspension concentrate, flowable liquid, and the like.
  • the inert carrier used in this invention may be either solid or liquid. Where the compound of the present invention is prepared into a solid formulation, appropriate additives and carriers may be incorporated with the compound.
  • the solid carrier may be a solid such as soybean flour, cereal flour, wood flour, bark flour, saw dust, powdered tobacco stalks, powdered walnut shells, bran, powdered cellulose, extraction residues of vegetables, powdered synthetic polymers or resins, clays (e.g. kaolin, bentonite, and acid clay), talcs (e.g. talc and pyrophyllite), silica powders or flakes (e.g.
  • diatomaceous earth silica sand, mica and white carbon
  • activated carbon powdered sulfur, powdered pumice, calcined diatomaceous earth, ground brick, fly ash, sand, calcium carbonate powder, calcium phosphate powder and other inorganic or mineral powders, chemical fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and ammonium chloride), and compost.
  • chemical fertilizers e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and ammonium chloride
  • compost may be used alone or as a mixture thereof.
  • an appropriate solvent may be used for dissolving or dispersing the compound in the liquid type formulation.
  • the liquid carrier is that which itself has solubility or which is without such solubility but is capable of dispersing an active ingredient with the aid of an adjuvant.
  • liquid carrier can be used alone or as a mixture thereof: water; alcohols such as methanol, ethanol, isopropanol, butanol and ethylene glycol; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, diisobutyl ketone and cyclohexanone; ethers such as ethyl ether, dioxane, dipropyl ether and tetrahydrofuran; aliphatic hydrocarbons such as kerosene and mineral oils; aromatic hydrocarbons such as benzene, toluene, xylene, solvent naphtha and alkylnaphthalenes; halogenated hydrocarbons such as dichloroethane, chloroform, carbon tetrachloride and chlorobenzene; esters such as ethyl acetate, diisopropyl phthalate, dibutyl phthalate
  • a surfactant is used.
  • examples of the surface active agent that can be added to the above-mentioned formulations include nonionic surface active agents, such as polyoxyethylene-added alkyl ether, polyoxyethylene-added higher fatty acid ester, polyoxyethylene-added sorbitan higher fatty acid ester and polyoxyethylene-added tristyryl phenyl ether, a sulfate ester of polyoxyethylene-added alkyl phenyl ether, an alkyl benzene sulfonate, a polycarbonate, a lignin sulfonate, a formaldehyde condensate of alkyl naphthalene sulfonate, and a copoly
  • adjuvants such as casein, gelatin, starch, methyl cellulose, carboxymethyl cellulose, gum arabic, polyvinyl alcohols, turpentine, bran oil, bentonite and ligninsulfonates.
  • adjuvants such as waxes, stearates and alkyl phosphates.
  • Adjuvants such as naphthalenesulfonic acid condensation products and polycondensates of phosphates may be used as a peptizer for dispersible products.
  • Adjuvants such as silicon oils may also be used as a defoaming agent.
  • adjuvants such as those described in U.S. Pat. No. 5,942,542 may be used.
  • the compound of the present invention may be used alone, it can be combined for the use with one or more of various types of other plant protection chemicals, for example, fungicides, insecticides, acaricides and synergists.
  • the composition may comprise a seed coating, formulated as described in U.S. Pat. No. 5,849,320.
  • the insecticide compounds of the present invention may be used in admixture with other agricultural and horticultural disease or pest controllers, acaricides, nematicides, bioagrochemicals, etc.; and herbicides, plant growth regulators, manures, etc. depending upon scenes using the present agricultural and horticultural insecticides, in order to expand both spectrum of controllable diseases and insect pest species and the period of time when effective applications are possible or to reduce the dosage.
  • the insecticide compounds of the present invention may be applied using a variety of protocols.
  • the composition may be applied to a crop on which the insect pests are expected to appear, or to a site where the appearance of the insect pests is undesirable.
  • the insecticide compositions of the present invention may also be applied to the plant seeds or the cultivation mediums for seeding such as soil to be seeded, the mat for raising seedlings, water, and the like, by the method of application to a nursery box, seed powdering, etc. or by the method of seed disinfection.
  • a plant absorb the compounds of the present invention by a seed treatment such as powder coating, dipping, etc., irrigation into seedling-raising carrier such as seedling-raising vessel, or planting hole, or by treatment of the culture solution for water culture.
  • a seed treatment such as powder coating, dipping, etc., irrigation into seedling-raising carrier such as seedling-raising vessel, or planting hole, or by treatment of the culture solution for water culture.
  • the applied dosage of the insecticide compounds of the present invention may be varied depending upon various factors such as the insect pests to be controlled, the growth state of a plant, weather, environmental conditions, the preparation form, application method, application site and application time.
  • the does may comprise a range of 0.1 g to 10 kg (in terms of the active ingredient) per 10 acres depending upon purposes.
  • the amount of an active ingredient in each of the composition may be in a range of from 0.01 to 90% by weight, or preferably from 0.05 to 85% by weight based on the total weight of the formulation.
  • the suitable content thereof is from 0.01 to 50% by weight.
  • Each of the prepared formulations such as wettable powder, emulsifiable concentrate, suspension concentrate and flowable solution, may be diluted with water or other solvent to be prepared and adjusted into the suspension or emulsion with a desired concentration to be applied to crop plants.
  • the formulations such as granular and dust formulations, the formulation itself is directly applied to the target crop plants or soil.
  • compositions of the present invention comprise compounds that increase FXR-mediated transcription and/or increase or potentiate EcR-mediated transcription present as non-toxic doses. JH levels in the insect support the use of FXR and EcR-activating compounds as insecticides.
  • the compounds may comprise a dosage ranging from 0.01 ⁇ M to about 10 mM, or from about 0.1 ⁇ M to about 1 mM, or from about 0.5 ⁇ M to about 50 ⁇ M.
  • JH III circulates in honeybee hemolymph at 0.5 ⁇ M (Elekonich, M. M. et al., 2001 , J. Insect Physiol., 47:1119-1125), which matches the concentration of the JH III precursor farnesyl diphosphate found in the rat liver (Bruenger, E. and H. C. Rilling, 1988 , Anal. Biochem., 173:321-327).
  • JH III titers in Diploptera hemolymph are 6 mM during the middle of the gonotrophic cycle and 10-fold lower at other times (To be, S. S., et al., 1985 , Experientia, 41:1028-1034).
  • CHO K1 cells were grown in Dulbecco's modified Eagle medium: nutrient mixture F-12 (1:1) containing 5% fetal bovine serum and supplemented with 50 u/ml penicillin, and 50 ⁇ g/ml streptomycin (Life Technologies) in a water-jacketed incubator held at 37° C. and maintained with a 5% CO 2 atmosphere.
  • Dulbecco's modified Eagle medium nutrient mixture F-12 (1:1) containing 5% fetal bovine serum and supplemented with 50 u/ml penicillin, and 50 ⁇ g/ml streptomycin (Life Technologies) in a water-jacketed incubator held at 37° C. and maintained with a 5% CO 2 atmosphere.
  • the precocene-like suicide substrate 3,4-dimethoxyisopentenylphenol was prepared as described Bowers, W. S., et al., 1976 , Science, 217:647-648) except that a 3,4-dimethoxylated reactant was used.
  • the transcriptional assay utilized for the study of EcR potentiation by JH was modified from the assay described by Forman et al., (Forman, B. M., et al., 1995, Cell 81, 687-695) as described below.
  • Cells were seeded in 6-well polypropylene culture plates (Falcon) with 10 5 cells per well on the day prior to transfection. Transfection was subsequently performed using either calcium phosphate (Kitareewan, S., et al., 1996, Mol. Biol. Cell, 7:1153-1196) or a GenePorter reagent (Gene Therapy Systems, Inc; San Diego, Calif.) following manufacturer's protocols.
  • the cells were incubated with plasmid DNA for seven hours and then washed with 1 ⁇ PBS.
  • Muristerone A (murA; Alexis Biochemicals) or 20-hydroxyecdysone (20E; Sigma) dissolved in ethanol to a concentration of 10 mM was diluted as necessary to the final assay concentration (FAC) in 2 ml of fresh incubation medium that was then applied to the cells.
  • FAC final assay concentration
  • JHIII Sigma
  • DMSO dimethyl sulfoxide
  • CDCA chenodeoxycholate
  • Transcriptional activity measured as 14 C-chloramphenicol counts (for measuring CAT activity) or relative luciferase activity (RLU) was quantified for each cell lysate. The counts were then normalized by adjusting for differences in ⁇ -galactosidase activity, since ⁇ -galactosidase expression is controlled by a constitutive promoter and provides an estimate of cell mass. Data were normalized as fold-induction based on differences in reporter gene activity between hormonally treated and control cells
  • CMX-EcR Drosophila EcR
  • CMX-USP Drosophila USP
  • CMX-FXR rat FXR
  • CMX-FXR human RXR ⁇
  • CMX-GR glucocorticoid receptor
  • CMX-GEcR glucocorticoid receptor trans-activating domain fused to an ecdysone DBD and LBD
  • the expression plasmids are constructed by inserting restriction fragments contining the appropriate coding sequence for the gene to be expressed into the CMXPL1 plasmid.
  • HRXR ⁇ an EcoRI fragment of human RXR- ⁇ (hRXR ⁇ ) was subcloned into the CMXPL1 plasmid.
  • the FXR expression vector contains DNA sequences encoding a constitutively active promoter derived from the Rous sarcoma virus long terminal repeat (LTR) fused to the complementary DNA sequence encoding the rat farnesoid-X-activated receptor (Forman et al., Cell 1995; NCBI Accession No. U18374) which is followed by a DNA sequence specifying the SV40 polyadenylation signal.
  • LTR Rous sarcoma virus long terminal repeat
  • pRS-rFXR was derived from pRS-hGR as follows: pRS-hGR was digested with Kpn I and Bam HI restriction endonucleases to release DNA sequences specifying the human glucocorticoid receptor (GR) and cDNA encoding rat FXR and its 5′ and 3′ untranslated sequences was then inserted into Kpn I and Bam HI digested pRS-hGR.
  • GR human glucocorticoid receptor
  • GGF comprising the glucocorticoid (G) amino terminus and DBD linked to the LBD of FXR
  • GGEc glucocorticoid
  • DBD glucocorticoid
  • the resulting plasmid was digested with Xho I and Bam HI and the GR-containing DNA fragment was isolated and ligated to an Xho I-Bam HI LBD fragment from an FXR variant prepared by hybridizing the oligonucleotide 5′-CTCGAGTGTATGTATACAGGTTTGTTAACTGAA-3′ (SEQ ID NO: 3) to another oligonucleotide 5′-AACAAACCTGTATACATACACTCGA-3′ (SEQ ID NO: 4) which was then ligated to a Hpa I-digested fragment from the pRSV-FXR expression vector.
  • the DBD/LBD junction in GGF is 5′- GMNLEARKTKKKIKGIQQATTGVSQ ECMYTGLLTEIQCKS-3′ (SEQ ID NO: 5) where the GR residues are underlined, amino acids GM are the last two residues of DNA binding domain and amino acids from FXR are in bold (not underlined).
  • the GGEc vector (Christopherson et al., 1992 , Proc. Natl. Acad. Sci., USA, 89:6314) is derived from the rat GR expression vector pRSV.GGG (Miesfeld et al., 1986, Cell, 46:389) and contains the Rous sarcoma virus LTR fused to DNA encoding the rat glucocorticoid receptor (GR) amino terminus and DNA binding domain fused to a DNA sequence encoding the Drosophila melanogaster ecdysteroid receptor (EcR) (NCBI Assession No. M74078; Koelle, M.
  • GEcEc i.e., GRdEcR
  • GEcEc was constructed by ligation of a Not I-Bam HI fragment containing the DBD and LBD of a modified EcR cDNA, EcR nx , in place of the DBD and LBD of the similarly modified GR expression vector construct pRShGR nx (Giguere et al., 1987 , Nature 330: 624).
  • the modified EcR cDNA was constructed using site-directed mutagenesis (Kunkel, 1985, Proc. Natl. Acad. Sci., USA, 82:488) to insert Not I (oligonucleotide template: 5′-CCTGCGCCACGGCGGCCGCCGGAGCTGTGCCTG-3′) (SEQ ID NO: 6) and Xho I (oligonucleotide template: 5′-GTGGGTATGCGCCTCGAGTGCGTCGTCCC-3′) (SEQ ID NO: 7) sites immediately flanking the DBD. This results in conversion of amino acids 259-261 from ValGlnGlu to ArgProPro and amino acid 331 from Pro to Leu.
  • Reporter plasmids EcR 5 - ⁇ MTV-LUC, and MMTV-LUC have also been described (Yao et al., 1992 , Cell, 71: 63-72; Yao et al., 1993 , Nature 366:476-478; and Forman, B. M., 1995 , Cell, 81: 687-693).
  • the reporter plasmids may be constructed by inserting a ecdysone reponse element (e.g., 5′-GATCCGACAAGGGTTCAATGCACTTGTCA-3′; SEQ ID NO: 8) at position-77 of a mouse mammary tumor virus (MMTV or MTV) promoter-reporter gene construct, such as MTV-CAT, MTV-LUC, MTV-GFP (Christopherson, K. S., 1992 , Proc. Natl. Acad. Sci., USA, 89: 6314-6318).
  • MMTV or MTV mouse mammary tumor virus
  • the (EcRE) 5 - ⁇ MTV-LUC construct was produced by subcloning the promoter region of MTV into the multiple cloning site of the p-LUC plasmid (Promega); this vector resembles the (EcRE) 5 ⁇ MTV-LUC described previously (No et al, Proc. Natl. Acad. Sci, USA, 93:3346-3351).
  • EcR-A, B1, and B2 sequences (Koelle et al., 1991 , Cell: 67, 59-77; Talbot et al., 1993 , Cell: 73:1323-1337; NCBI Accession Nos. S63761 and S63762) generated by PCR were cloned into the BamHI and XbaI sites of the pcDNA3 vector (InVitrogen).
  • the EcRA DNA fragment was produced by using PCR amplification of EcRA DNA with the pWT57 vector as a template (Talbot et al, 1993).
  • the forward primer, DEAf (5′-CACCCGGATCCACCATGTTGACGACGAGTGGACAA) (SEQ ID NO: 9) was used with the reverse primer, DEr (5′-ACCTCTCTAGACTATGCAGTCGTCGAGTGGTC) (SEQ ID NO: 10) to produce a fragment that was subsequently digested with BamHI and XbaI and ligated into a pcDNA3 vector digested with BamHI and XbaI.
  • the plasmid encodes a version of EcRA that includes 849 amino acids, and deletes the F domain.
  • the EcRB1 DNA fragment was produced by using PCR amplification with the pMK1 vector (Koelle et al, 1991; Talbot et al, 1993) using the aforementioned DEr primer with the forward primer, DEB1f (5′-CACCCGGATCCACCATGAAGCGGCGCTGGTCGAAC) (SEQ ID NO: 11). The fragment was subsequently digested and cloned into the pcDNA3 vector as described for A. The plasmid encodes a version of EcRB1 that includes 878 amino acids, and deletes the F domain.
  • the EcRB2 DNA fragment was produced by using PCR amplification with the pWT56 vector (Talbot et al, 1993) using the aforementioned DEr primer with the forward primer, DEB2f (5′-CACCCGGATCCACCATGGATACTTGTGGATTAGTA-3′) (SEQ ID NO: 12). The fragment was subsequently digested and cloned into the pcDNA3 vector as described for A.
  • the plasmid encodes a version of EcRB2 that includes 669 amino acids, and deletes the F domain.
  • the VP16dEcR vector has also been described previously (Yao, T. P, et al., 1993, Nature 366: 476-479; No, D., et al., 1996 , Proc. Natl. Acad. Sci USA, 93:3346-3351; Mouillet et al., 2001; Henrich et al., 2003).
  • the VP16 EcR vector was constructed using PCR amplification with pWT57 with the reverse primer, Der (described above), together with the forward primer Def (5′-CACCCGGATCCACCATGAAGAAGGGACCTGCGCCA-3′) (SEQ ID NO: 13).
  • the fragment was subsequently digested with BamHI and XbaI and cloned in to the multiple cloning site of pVP16 (Clontech).
  • the resulting vector encodes a protein consisting of the VP16 activation domain linked to the C-E domain of EcR and consisting of 626 amino acids.
  • the VP16CfUSP vector is also previously described (Palli, SR, et al., 2003 , Eur J. Biochem, 270:1308-15).
  • the USP vector contains the USP LBD of Choristoneura fumeriferana (NCBI Accession No. AF045891). Both VP16 chimeras contain an N-terminal domain that is active in mammalian cells (Louvion et al, 1993 , Gene: 131:120-134).
  • PCR amplification with a forward primer including an EcoRI site and a reverse primer including a BamHI site was used to produce a fragment that encodes the D-F domains of Choristoneura fumiferana USP (Accession #AAC31795).
  • the resulting PCR product was digested with EcoRI and BamHI and cloned into the pVP16 vector (Clontech) to produce the fusion protein.
  • VP16-USP fusion vectors VP16-dUSPF1, F2, and F3, have been generated. These vectors were constructed using the pZ7-1 vector (Henrich et al, Nucl. Acids Res, 18:4143-4148, 1990) encoding Drosophila melanogaster USP. F1 was constructed with the forward primer 5′-TTTTGAATTCAGCGGCAGCAAGCACCTCTGC-3′ (SEQ ID NO: 14) together with the reverse primer, 5′-TTTTAAGCTTTAGAGTCGGGACCCTACTCC-3′ (SEQ ID NO: 15).
  • the resulting PCR product was digested with EcoRI and HindIII and cloned into pVP16 (Clontech) digested with EcoR1 and HindIII.
  • the resulting protein encodes a VP16 activation domain fused to the last six amino acids of the A/B domain and the C-E domains of USP.
  • F2 was constructed using the same pZ7-1 vector, reverse primer and the forward primer (5′-TTTTGAATTCTGCTCTATTTGCGGGGATCGG-3′) (SEQ ID NO: 16).
  • the resulting PCR product was cloned into VP16 using the same approach described for F1.
  • the resulting protein encodes a VP16 activation domain fused to the C-E domain of Drosophila melanogaster USP.
  • F3 was constructed as both F1 and F2 except that the forward primer used was (5′-TTTTGAATTCAAGCGCGAAGCGGTCCAGGAG′3′) (SEQ ID NO: 17).
  • the resulting protein encodes a VP16 activation domain fused to the D-E domain of Drosophila melanogaster USP.
  • a GRdEcR chimera that consists of the rat glucocorticoid receptor (GR) activation domain attached to the EcR DBD and LBD ( FIG. 1 ) was cotransfected along with mRXR ⁇ into CHO cells.
  • the response of transfected cells to murA was measured using a (EcRE) 5 - ⁇ MTV-CAT reporter plasmid that carries five tandem repeats of the hsp27 EcRE linked to the MTV (mouse mammary tumor virus) promoter and the chloramphenicol acetyltransferase gene (CAT).
  • EcRE 5 - ⁇ MTV-CAT reporter plasmid that carries five tandem repeats of the hsp27 EcRE linked to the MTV (mouse mammary tumor virus) promoter and the chloramphenicol acetyltransferase gene (CAT).
  • FIG. 2 Results of a typical experiment are shown in FIG. 2 .
  • Cotransfection with GrdEcR and RXR evoked a detectable response at dosages as low as 0.1 ⁇ M murA.
  • juvenile hormone III JHIII
  • JHIII juvenile hormone III
  • FIG. 2 sets 2 and 3, respectively.
  • JHIII did not display the ability to evoke a response that was greater than the maximal level induced by 10 ⁇ M murA.
  • the potentiation experiments were repeated with a chimera encoding the VP16 activation domain connected to the DBD and LBD of Drosophila EcR to generate the construct VP16dEcR ( FIG. 3 ).
  • Luciferase was used as a reporter gene, (EcRE) 5 - ⁇ MTV-LUC. All activities are normalized fold-inductions in relative light units from cells incubated with hormone for 20 hours compared to luciferase activity levels in cells incubated with solvents only. For all combinations, 80 ⁇ M JHIII was used. The dosage of muristerone A (murA) used was based upon preliminary experiments to determine a submaximal dosage wherein a JHIII effect, if any, was detectable. All data are based on the mean normalized fold-inductions from at least three replicates. The range (coefficient of variation) of fold-inductions was less than 15% among replicates.
  • VP16dEcR tested with muristerone A generated a sensitive and robust ecdysteroid response (based on normalized fold-induction).
  • the VP16dEcR partnered with mouse RXR (mRXR), showed a response to 0.01 ⁇ M and 0.1 ⁇ M muristerone A that was further potentiated by JHIII ( FIGS. 3 a and 3 b , respectively).
  • the VP16dEcR chimera also displayed a discernible response to 20E at 10 ⁇ M (over 20-fold) using RXR.
  • the VP16dEcR/20E activity was only minimally affected by the additional presence of JHIII, however (not shown).
  • FIG. 3 c shows results at 0.1 ⁇ M murA.
  • the same degree of potentiation was observed using the VP16dEcR/USP combination as with the VP16dEcR/RXR when using murA, except that a higher murA dose was required to achieve the same efficacy.
  • the normalized level of JHIII-mediated potentiation of the murA response with USP was similar to that seen for RXR.
  • the combination of VP16dEcR with VP16CfUSP combination showed no response to 10 ⁇ M 20E and was not affected by the additional presence of JHIII (data not shown).
  • each of the three natural Drosophila melanogaster EcR isoforms (EcRA, EcRB1, and EcRB2) were cotransfected into CHO cells with the VP16CfUSP fusion protein. Results are shown in FIG. 3 , sets d-f. Again, for all combinations, 80 ⁇ M JHIII was used, and the dosage of muristerone A (murA) used was based upon preliminary experiments to determine a submaximal dosage wherein a JHIII effect, if any, was detectable. For the results shown in FIG. 3 , all data are based on the mean normalized fold-inductions from at least three replicates, except for EcRA (two replicates).
  • EcR isoforms are known to differ at the N-terminal region of the protein, which is the part of the protein involved in dimerization of EcR with either USP, RXR or other appropriate partners. All three isoforms showed some transcriptional capability in the mammalian cell culture system. Interestingly, however, none of the isoform constructs showed the same dosage sensitivity seen with VP16dEcR/RXR (direct comparison not shown).
  • the EcRB1/VP16CfRSP combination when tested in the absence of hormone, the EcRB1/VP16CfRSP combination showed a relatively high level of ligand-independent transcription.
  • the EcRB1/VP16CfUSP had between 10 and 20-fold higher basal levels than any other EcR construct tested.
  • the EcRA isoform in combination with VP16CfUSP also showed a level of basal level of transcription that was 2 to 3 fold higher than that observed for EcR-B2.
  • the basal activity of the B2/VP16CfUSP dimer was about the same as the basal activities produced by the VP16dEcR/RXR and GEcEc/RXR (i.e., GRdEcR/RXR).
  • FIG. 4 shows the effects of murA, 20E, and JHIII on RLU activity induced by (EcRE) 5 ⁇ MTV-LUC in CHO cells cotransfected with a Drosophila EcR isoform and VP16CfUSP where sets 1, 2, and 3 in the figure correspond to EcRA, EcRB1 and EcRB2, respectively.
  • EcRE EcRE
  • VP16CfRSP all three Drosophila isoforms were induced by about 30-40 fold at 1 ⁇ M murA.
  • the response of all EcR isoforms in the presence of VP16CfUSP was potentiated by the presence of 80 ⁇ M JHIII in the presence of 0.1 ⁇ M murA ( FIG. 4 ). It was found that the effect was dose-dependent (data not shown) similar to the results seen with GEcEc (i.e, GRdEcR).
  • the range of the normalized fold inductions for each experiment was found to vary by less than 15% for each experiment.
  • the Drosophila EcR isoforms and VP16CfUSP were also tested with 20E ( FIG. 4 ). At a dosage of 10 ⁇ M 20E, all three constructs generated a consistent and discernible transcriptional response. Only the EcRB2/VP16CfUSP dimer ( FIG. 4 , set 3) was potentiated significantly by the additional presence of JHIII, however. That only the B2 EcR isoform is potentiated by JHIII in the presence of 20E indicates that JHIII potentiation may depend upon both the N-terminal domain of EcR and the activating ecdysteroid.
  • the activity observed among the three EcR isoforms was dependent upon the identity of the heterologous partner used. For example, in contrast to the robust response observed when RXR was tested with VP16dEcR, RXR did not mediate a response to murA as a dimer with either EcRA or EcB2. Only EcRB1/RXR displayed a response to murA among the three isoforms ( FIG. 3 d, e , and f), but the levels of transcription were relatively low and dramatically reduced from those noted for the B1/USP combination. The murA response was further potentiated by JHIII with the B1/RXR combination, though JHIII by itself failed to evoke any response. Unlike the 20E response noted for all the isoforms with USP as a partner, none of the isoforms showed a 20E response with RXR as a heterodimeric partner. Absolute transcription levels were also relatively low with RXR.
  • VP16 and RXR, B2 and USP result in a functional receptor that is capable of showing an ecdysteroid response and/or JHIII potentiation.
  • Levels of ligand-independent transcription also depend upon both the EcR N-terminal domain and the heterodimeric partner.
  • the potentiation observed in the experiments cannot be attributed to the activation of RXR by either JHIII or a JHIII metabolite, since JHIII showed no activity by itself on the assays (Harmon, M. A., et al., 1995 , Proc. Natl. Acad. Sci. USA, 92:6157-6160; Saez, E., et al., 2000 , Proc. Natl. Acad. Sci. USA, 97:14512-14517).
  • candidate juvenoids were tested for their ability to activate FXR mediated transcription by transfecting Chinese hamster ovary (CHO) cells using the FXR plasmid, mouse RXR ⁇ , and the ⁇ MTV-(EcRE) 5 -CAT reporter as described herein.
  • An RXR-dependent CRBPII-CAT reporter plasmid was employed in parallel assays to discern activators specific for this receptor.
  • FIG. 6 shows the metabolic route from farnesyl diphosphate (FDP) to methyl farnesoate in mammals or juvenile hormone III in insects and the relative efficacy of each isoprenoid as an inducer of FXR-dependent transcription.
  • FDP farnesyl diphosphate
  • a negative sign indicates no activity and positive signs correlate with the efficacy (ratio of maximal inducible activity achieved with the highest non-cytotoxic dose of activator compared to that obtained with vehicle).
  • Transcriptional activities ranged from 2-fold increases for farnesoic acid to 20-fold inductions for juvenile hormone III when tested at 50 ⁇ M.
  • Alcohol and aldehyde dehydrogenases oxidize farnesol farnesal and farnesoic acid (Christophe, J. and G Popjak, 1961 , J Lipid Res., 2:244-257), which exhibited 2 to 3-fold activity increases in FXR mediated transcriptional activity, respectively.
  • Insects and mammals transform farnesoic acid into methyl farnesoate (Schooley, D. A. and F C Baker, 1985, Juvenile hormone biosynthesis.
  • FXR-activating farnesoids have been described as JH agonists in insect bioassays (Schneiderman, H. A., and L I Gilbert, 1964 , Science, 143:325-333). Like farnesol, nerolidol was effective as a juvenoid and as an FXR effector (Table 4). The chlorophyll metabolite phytol increased activity three times, thereby evincing marginal JH activity (Table 4). In contrast, neither the monoterpenes linalool (200 ⁇ M) nor geraniol were effective as FXR activators or juvenoids.
  • Synthetic juvenoids (Table 4 and FIG. 7A ) were also evaluated.
  • the synthetic juvenoids, methoprene and pyriproxyfen increased FXR-dependent activity with efficacies like that produced by JH III (Table 4 and FIG. 7A ).
  • FXR is Activated by Insecticide Synergists
  • FXR effectors For example, cedarwood oil and its redolent sesquiterpene ⁇ - and ⁇ -ionone isomers increased FXR-dependent activity with potencies and efficacies like those induced by farnesol (Table 5). It is known that sesamin blocks liver HMG CoA reductase activity, that farnesol is anti-proliferative, and that FXR is inhibited by the hypocholesterolemia-inducing plant steroid guggulsterone (Wu, J. et al., 2002 , Mol Endocrinol., 16:1590-7; Urizar, N. L.
  • Limonene in orange oil blocks tumors, inhibits HMG CoA reductase, and is being tested as a human chemotherapeutic agent (Crowell, P. L. and M. N. Gould, 1994 , Crit. Rev. Oncog., 5:1-22; Elegbede, J. A. et al., 1984, Carcinogenesis, 5:661-4.; and McNamee, D., 1993, Lancet 342, 801).
  • Limonene and limonene oxide (400 ⁇ M) were inactive in activation of FXR, but either enantiomer of its metabolite perillyl alcohol induced FXR-dependent CAT activity 4-fold (Table 5). This dose matches that required to inhibit cell growth in culture (He, L. et al., 1997 , J. Nutr., 127:668-674). Finally, while menthol and fenchone (fennel) were inactive (0.5 mM), 0.5 mM fenchyl alcohol induced activity 5-fold, as did 0.5 mM pine tree-derived pinane diol (Table 5).
  • Forskolin one of the most robust FXR effectors yet described (Howard, W. C. et al., 2000 , Tox. Appl. Pharm., 163:195-202), increased activity more than 100-fold when added at 2 ⁇ M.
  • 1-trans- ⁇ 9 -tetrahydrocannabinol (THC) from the cannabis plant contains a tricyclic ring.
  • cannabinoids block cell growth, inhibit DNA synthesis, and lower the incidence of spontaneously-arising mouse liver tumors (Carchman, R. A., et al, 1976 , Cancer Res., 36:95-100; Munson, A. E.
  • Phorbol-like diterpenes from croton tiglium and related plants such as phorbol 12,13-dibutyrate, mezerein, and ingenol 3,20-dibenzoate, increased activity 2, 3, and 5-fold, respectively, when added at 10 ⁇ M (Table 5). Phorbol and ingenol were inactive.
  • Ursolic acid is a rosemary ingredient related to oleanolic acid that inhibits mouse skin tumors (Huang, M. T. et al., 1994 , Cancer Res., 54:701-708; Nishino, H. et al., 1988 , Cancer Res., 48:5210-5215). While rosemary oil maximally induced FXR-dependent activity 12-fold (Table 5), ursolic acid (50 ⁇ M) was ineffective as were its polyketide constituent rosemarinic acid and its curcumin congener.
  • Cucurbitacins are phytoecdysteroids that inhibit ecdysone receptor (EcR) function (Dinan, L. et al., 1997 , Biochem J, 327:643-50).
  • EcR ecdysone receptor
  • Cucurbitacin D (1 ⁇ M) suppressed FXR-dependent activity promoted by JH III and CDCA (40 ⁇ M each), 7- and 58-fold, respectively ( FIG. 8C ).
  • the ⁇ 1 -unsaturated congener cucurbitacin I also inhibited farnesol-induced activity with an IC 50 ⁇ 50 nM (data not shown).
  • the 22-oxo- ⁇ 23 -ene group of cucurbitacin is critical for EcR antagonist activity, allegedly by forming covalent adducts between its ⁇ , ⁇ -unsaturated carbonyl group and amino acids in the EcR ligand binding domain (Dinan, L. et al., 1997 , Biochem J, 327:643-50).
  • 22R-Hydroxycholesterol is metabolized in mammals to pregnenolone via a 20,22-dihydroxycholesterol intermediate with an apparent K m of 7 ⁇ M (Sugano, S., et al., 1966, J Biochem (Tokyo) 1996, 120:780-7).
  • methylenedioxyphenyls like myristicin and apiole in dill and parsley weakly elevated CAT activity 4-fold at 250 ⁇ M, but their congener safrole, a rodent liver carcinogen (Miller, E. C. et al., 1983 , Cancer Res., 43:1124-34), was inert ( FIG. 8E ). While the alkenylbenzenes eugenol and caffeic acid were also inactive, methyleugenol, a multi-site rodent carcinogen (Johnson, J. D., et al.,. J. Agric. Food Chem. 2000, 48:3620-3632) found in nutmeg and other plants, induced FXR-dependent activity 4 times more than vehicle at this same dose ( FIG. 8E ).
  • Silybin increased FXR-dependent activity 18-fold at 50 ⁇ M (Table 5). While silymarin lowers cholesterol better than silybin (Krecman, V. et al., 1998 , Planta Med., 64:138-42), FXR activity was maximally increased only 4-fold by the former (data not shown). FXR may respond to other analogs of silybin in milk thistle such as silydianin, silychristin or their metabolites. Taxifolin is one silymarin component that has been shown to inhibit HMG CoA reductase activity in hepatocytes (Theriault, A., et al., 2000 , J. Lipid Res., 41:1969-1979).
  • FXR was unresponsive to taxifolin or to other flavonoids such as genistein, quercetin, catechins, and gossypetin (50 ⁇ M). However, activity was increased 4-fold in response to the same dose of tangeretin, a methoxylated flavone in citrus fruits.
  • metabolites of rotenone such as 6′,7′-dihydro-6′,7′-dihydroxyrotenone and 8′-hydroxyrotenone, which are hydroxylated in the proximity of the furan ring, can be generated using microsomal homogenates prepared from either insect or rodent tissues (Fukami, J. I. et al., 1967 , Science, 155:713-6).
  • xiii Xanthines FXR activation by forskolin animated tests of other plant compounds that modulate cAMP levels. FXR-dependent activity was induced 12-fold by 3 mM theophylline or caffeine, but congeners such as theobromine, hypoxanthine, xanthine, adenine, and the cAMP metabolite 5′-AMP were ineffective ( FIG. 8H and data not shown). Modest (4-fold) increases in CAT activity were afforded by 8-Br-cAMP or dibutyryl cAMP (1 mM).
  • FXR activation by plant-derived JHs and secondary substances provoked tests of man-made insecticides. Its sodium channel-based toxicity notwithstanding (Soderlund, D. M.,: 1985 , Neurotoxicology, 6:35-46), a pyrethrum extract of chysanthemum flowers maximally induced FXR-dependent activity 13-fold.
  • Candidate active ingredients are the structurally-related cinerins, pyrethrins, and jasmolins since FXR was induced by synthetic pyrethroids (25 ⁇ M) including cypermethrin (15-fold induction), permethrin (5-fold induction), phenothrin (8-fold induction), and bioallethrin (14-fold induction) (Table 6).
  • Organochlorine insecticides such as o,p-DDT (but not p,p-DDT), chlordane, kepone, lindane, dieldrin, and toxaphenes increased CAT activity 3, 7, 12, 5, 17, and 9-fold, respectively (data not shown).
  • Organochlorines like aroclor 1254 (5 ⁇ M) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (100 nM) increased activity 5 and 15-fold, respectively (data not shown).
  • FXR was activated by organophosphates such as malathion, diazinon, chlorpyrifos, and parathion (25 ⁇ M) with potencies and efficacies like farnesol (Table 6). Others like ethion and coumaphos were more efficacious and more potent, exhibiting 50- and 16-fold increases in activity, respectively, when tested at 5 ⁇ M. Lower molecular weight insecticides phosdrin, carbaryl, and imidan were inactive. Phenylpyrazoles such as chlorfenapyr which lowers ATP levels and structurally-related fipronil that distinctively blocks chloride channels increased FXR-dependent activity 10- and 20-fold, respectively, at 25 ⁇ M ( FIG. 9 ).
  • FXR was unaffected by imidacloprid, a nicotine-like compound that interferes with acetylcholine receptor function. These results indicate that FXR may activate transcription in response to structurally-diverse synthetic chemicals that manifest pleiotropic cytotoxicities through equally disparate mechanisms.
  • F. FXR May be Inhibited or Activated by a Metabolite of the JH Antagonist Precocene
  • Chromene ring-containing precocenes are plant-derived JH antagonists that hasten insect metamorphosis (Bowers, W. S. et al., 1976 , Science, 193:542-7). Like safrole and other alkenylbenzenes, the precocenes are rodent hepatocarcinogens (Wiseman R. W. et al., 1987 , Cancer Res., 47:2275-83). Their alkylation of DNA and proteins and their metabolism to 3,4-diols hinted that precocenes may form reactive epoxides (Brooks, G. T., et al., 1979 , Nature, 281:570-572; Pratt, G.
  • Precocene I (25 mg, Sigma-Aldrich, 99% purity) was separated by thin layer chromatography using hexane-ethyl acetate (9:1) as the mobile phase. Aliquots (1 ⁇ and 3 ⁇ doses) were tested for FXR effector activity in parallel.
  • precocene carbons are numbered for reference, and the putative structure for the precocene dimer is presented based upon FXR responsiveness (box within graph).
  • One prospective silica gel-catalyzed air oxidation product of precocene (3,4-dihydroxyprecocene) is depicted.
  • FIG. 10C shows that precocene I and precocene II are metabolically interconverted by methylations and demethylations; also shown is a model (in box) for oxidation (O) and tautomerization of 3,4-dimethoxy-6-isopentenylphenol to a quinone methide and subsequent adduct formation with cellular nucleophiles.
  • Ubiquinone-1 (U1) with a single isoprene unit is a congener of decaprenylated U10 (coenzyme Q), which functions in electron transport.
  • U1 completely inhibited farnesol-induced FXR-dependent activity ( FIG. 1E ).
  • di-, tri-, and tetraprenylated ubiquinones (U2, U3, and U4) increased activity 7, 5, and 3-fold, respectively (data not shown).
  • FXR activation by U2 is depicted ( FIG. 10F ).
  • U6, U8, U9, and U10 were inactive (data not shown).
  • U2, U3, and U4 are detected in bacteria and U6 is found in yeast (Daves, G. D. et al, 1967 , Biochemistry, 6:2861-2866). However, only U9 and U10 have been reported in insects and mammals (Olson, R. E., 1966 , Vitam Horm., 24:551-74). U1, U2, U3, and U4 have not been measured in mammals and hence their physiological significance is not known. Nonetheless, these results illustrate how in situ-generated electrophilic metabolites of precocene may antagonize the effects of JHs via interactions with an FXR homolog in insects.
  • FXR may have pharmacological features of a long-postulated insect JH receptor.
  • the prominent candidate for this JH-responsive macromolecule in insects is the structurally-related ecdysone receptor (EcR).
  • EcR ecdysone receptor
  • EcR heterodimerizes with RXR and binds to the hsp27 ecdysone-responsive DNA element (Forman et al., 1995), but distinctively activates transcription in response to muristerone A (MurA or MUR), a synthetic ecdysone (Yao, T. P., et al., 1992) Cell, 71:63-72).
  • GEcEc MurA-inducible chimeric receptor
  • GR human glucocorticoid receptor
  • FIG. 11A failed to respond to 80 ⁇ M JH III ( FIG. 11A ).
  • muristerone A MUR
  • JH III (12, 25, or 50 ⁇ M) were added (underlying triangles). Numbers over bars indicate the ratio of the GEcEc-dependent activity produced by 50 ⁇ M JH III in the presence of the indicated dose of MUR to the activity produced by MUR alone.
  • Relative light units (RLU) from firefly luciferase in cell lysates are expressed relative to renilla luciferase (Promega).
  • CHO cells were separately transfected with plasmid DNAs that express mouse RXR ⁇ or GEcEc (1.25 ⁇ g plasmid DNA per well), or transfected with both plasmids or none.
  • MUR was added to cells at 10 nM and JH III at 50 ⁇ M.
  • Normalized CAT activity was determined by measuring ⁇ -galactosidase activity produced by cotransfected SV40- ⁇ -gal plasmid DNA. Note that the ordinate axis is broken. The potentiative effect was also seen using a VP16- Chironomus Usp substituted for its mammalian homolog RXR (data not shown). Also, both RXR and GEcEc were essential for activity ( 11 B).
  • GEcEc-dependent activity was induced between two and three times by other insecticides (25 ⁇ M) including diazinon, endosulfan (5 ⁇ M), coumaphos, permethrin, and precocene, while 25 ⁇ M chlorpyrifos potentiated the MUR response nine times ( FIG. 11D ).
  • insecticides 25 ⁇ M
  • coumaphos coumaphos
  • permethrin coumaphos
  • precocene 25 ⁇ M chlorpyrifos potentiated the MUR response nine times
  • FIG. 11D triplicate wells of CHO cells were transfected with GEcEc and RXR, after which muristerone A (MUR) was added at 10 nM in ethanol vehicle. Cells were incubated with the indicated natural and synthetic insecticides (25 ⁇ M), except endosulfan which was added at 5 ⁇ M. Plus and negative signs refer to MUR addition
  • FXR and EcR exhibit substantial homology, FXR was unresponsive to muristerone A, ecdysone, or 20-hydroxyecdysone. Reciprocally, the FXR activator chenodeoxycholic acid (CDCA) did not induce GEcEc (data not shown). Thus, it appears that EcR and FXR may both activate transcription in response to natural and synthetic JHs and insecticides, but that the two receptors may not be seamlessly interchangeable.
  • LBDs The ligand binding domains (LBDs) of nuclear receptors map to their carboxyl termini (Kumar, R. and E. B. Thompson, 1999 , Steroids, 64:310-9).
  • DBD GR DNA binding domain
  • luciferase reporter construct was used.
  • results were normalized using a reporter plasmid from which CAT expression is driven by a minimal promoter consisting of dimerized GREs linked upstream of a 13 base pair TATA box DNA element derived from the adenovirus E1B gene.
  • GGEc and RXR were transfected into CHO cells with either a CAT or a luciferase reporter plasmid containing 1.5 kilobase pairs of the glucocorticoid-inducible mouse mammary tumor virus (MTV) promoter.
  • MTV mouse mammary tumor virus
  • Farnesol was added at 25 ⁇ M with or without 0.2 ⁇ M muristerone A.
  • Normalized CAT activities are mean ⁇ standard deviation as measured from lysates sampled from triplicate wells. Like GEcEc, this activity required an RXR-expressing plasmid (data not shown).
  • GGEc responded to juvocimene (20 ⁇ M), the JH mimetic from basil, by increasing activity 7 times more than MUR ( FIG. 12B ).
  • CHO cells were transfected with a GGEc-expressing plasmid, a mouse RXR ⁇ expression plasmid, and an adenovirus E1b TATA-(GRE) 2 -CAT reporter plasmid.
  • the GGEc plasmid was eliminated from DNA mixtures added to cells transfected in parallel.
  • CAT activities were measured from lysates of cells from triplicate wells treated with ethanol vehicle, juvocimene (20 ⁇ M), muristerone A (0.2 ⁇ M), or both.
  • a plasmid was constructed that expresses a hybrid protein (GGF) consisting of the human GR amino terminus and DBD linked to the carboxyl terminus of FXR. GGF-dependent transcriptional activity was increased 3- and 15-fold by farnesol and CDCA (40 ⁇ M each), respectively ( FIG. 12D ). The limited GGF-dependent induction by farnesol was doubled by cotransfecting an RXR-expressing plasmid ( FIG. 12D ). Importantly, no activity was detectable without GGF.
  • GGF hybrid protein
  • this FXR-like responsiveness led to tests of natural and synthetic insecticides as GGF effectors.
  • CHO cells were transfected with receptor plasmids expressing GGF and mouse RXR ⁇ and a luciferase reporter plasmid containing the upstream 1.5 kbp of the MTV promoter.
  • GGF-dependent activity was stimulated between 2- and 7-fold in response to micromolar doses (e.g., 25 ⁇ M) of cypermethrin, diazinon, dieldrin, fipronil, piperonyl butoxide, rotenonic acid, cafestol, precocene, methyl jasmonate, and abietic acid ( FIG.

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